Kκ/μ-like protein tyrosine phosphatase, PTP λ

ABSTRACT

This invention concerns novel receptor protein tyrosine phosphatase polypeptides. Specifically, this invention concerns the novel receptor protein tyrosine phosphatase λ which is related to the homotypically adhering receptor protein tyrosine phosphatases κ and μ. The invention further relates to analogs of these polypeptides in other mammals, functional derivatives thereof, antibodies which are capable of specifically binding to these polypeptides, nucleic acids encoding these polypeptides, vectors containing and capable of expressing such nucleic acid and recombinant host cells transformed with such nucleic acid. Methods for the recombinant production of these receptor protein tyrosine phosphatase polypeptides and assays for identifying agonists and antagonists of these polypeptides are also within the scope of the invention.

This is a division of application Ser. No. 08/652,971 filed May 24, 1996now U.S. Pat. No. 5,814,507.

FIELD OF THE INVENTION

This present invention concerns novel receptor protein tyrosinephosphatase polypeptides. More particularly, the present inventionconcerns a novel receptor protein tyrosine phosphatase designated hereinas PTP λ.

BACKGROUND OF THE INVENTION

An extraordinary number of cellular processes are regulated by thetyrosine phosphorylation of a diversity of proteins. Tyrosinephosphorylation is induced by a plethora of receptor-like molecules aswell as by a wide range of intracellular enzymes. The effects oftyrosine phosphorylation are numerous, and they modulate a range ofdevelopmental as well as other cellular operations. Of course, theimportance of tyrosine phosphorylation is underlined by the need formechanisms which carefully regulate the levels of these events. Thus,protein tyrosine kinases represent positive mediators of tyrosinephosphorylation, while protein tyrosine phosphatases (PTPs) induce theremoval of phosphate from tyrbsine. The balance of the levels oftyrosine phosphate is thus mediated by the relative activities of thesetwo types of enzymes. It is therefore clear that the mechanisms whichregulate cellular function via tyrosine phosphorylation require specificproteins which mediate both the upregulation as well as thedownregulation of the levels of this modified amino acid.

PTPs represent a growing family of enzymes that are found in bothreceptor as well as non-receptor forms (Tonks, Semin. Cell. Biol.4:373-453 (1993), Walton et al., Ann. Rev. Biochem. 62:101-120 (1993)and Sun et al., Trends Biochem. Sci. 19(11):480-485 (1994)).Non-receptor PTPs are a highly diverse kindred, and they contain anumber of motifs, in addition to the enzymatically active PTP domain,that serve to regulate the region of the cell occupied by these proteinsas well as the substrate specificity of these enzymes. The receptor PTPsare also a highly diverse group that are unified by the inclusion of atransmembrane domain which disposes them to the plasma membrane of thecell. Recently, the receptor PTPs have been subdivided into 8 typesbased upon their domain content (Brady-Kalnay et al., Curr. Opin. Cell.Biol. 7(5):650-657 (1995)). These subtypes all contain one or two PTPasedomains on their cytoplasmic sides, with a variety of extracellularmotifs including heavily O-glycosylated mucin-like domains (for example,CD45), chondroitin sulfate domains (for example, PTP γ) and short,highly glycosylated segments (for example, PTP α). The largest family ofPTPs is the family which contains various motifs related to those foundin adhesion molecules. These motifs include immunoglobulin-like (IgG)domains and fibronectin type III (FnIII) regions similar to those foundin cell adhesion molecules such as ICAM, N-CAM and Ng-CAM (Rao et al.,J. Cell. Biol. 118:937-949 (1992)). In addition, a subset of theseadhesion-like PTPs, including the PTPs κ and μ, contain a third domaintermed the MAM, for meprin/A5/PTP μ, motif (Beckman et al., TrendsBiochem. Sci. 18:40-41 (1993)). The MAM motif has been previously shownto be involved with cell-cell recognition in neurons (Jiang et al., J.Biol. Chem. 267:9185-9193 (1992), Takagi et al., Neuron 7:295-307 (1991)and Hirata et al., Neurosci. Res. 17:159-169 (1993)). Interestingly,recent data suggest that three of these adhesion-like PTPs appear to beinvolved with neuronal pathfinding during Drosophila development (Desaiet al., Cell 84:599-609 (1996) and Kreuger et al., Cell 84:611-622(1996)). Together, these structural data are consistent with theconjecture that receptor PTPs encompass a diverse family ofenzymatically active proteins which contain a number of interesting cellsurface motifs potentially involved with the sensing of theextracellular environment.

PTPs κ and μ are the receptors that are most well characterized asadhesion molecules (Brady-Kalnay et al., supra, Jiang et al., Mol. Cell.Biol., 13:2942-2951 (1993) and Gebbink et al., Febs. Lett.290(1-2):123-130 (1991)). Both of these PTPs have been demonstrated tomediate homotypic adhesion. Thus, a diversity of assays, including cell-as well as molecule-based, have demonstrated that the extracellulardomain of these enzymes can bind with high specificity in a homophilicmanner (Brady-Kalnay et al., J. Cell. Biol. 268:961-972 (1993), Gebbinket al., J. Biol. Chem. 268:16101-16104 (1993) and Sap et al., Mol. Cell.Biol. 14:1-9 (1994)). Interestingly, mixing experiments have revealedthat these closely related PTPs will not bind to each other in aheterophilic mode, suggesting that the extracellular domain is meant torecognize other cells specifically expressing identical receptors, asituation highly reminiscent of the cadherin homotypic adhesion system(Kemler et al., Trends Genet. 9:317-321 (1993)). While the extracellulardomains required for this homotypic binding remain controversial, itappears likely that both the MAM motif as well as the IgG region areinvolved with homophilic interactions (Brady-Kalnay et al., J. Biol.Chem. 269:28472-28477 (1994) and Zondag et al., J. Biol. Chem.270(24):14247-14250 (1995)). While these data suggest that thesehomophilic adhesion enzymes are involved with the recognition of othercells expressing similar types of receptors, other data have suggestedthat this recognition event may play a role in the attachment of suchcells to each other. Thus, Tonks and colleagues have recentlydemonstrated that the receptor PTP μ specifically associates with thecatenin/cadherin complex of homotypic cell adhesion molecules(Brady-Kalnay et al., J. Cell. Biol. 130(4):977-986 (1995)). They alsodemonstrated that treatment of cells with the PTP inhibitor pervanadateresulted in the upregulation of tyrosine phosphorylation of cadherinsand catenins, a result which suggested a role for a PTP, potentially PTPμ, in the maintenance of the cadherin/catenin complex in anunderphosphorylated state. Interestingly, previous work suggested thatthe level of tyrosine phosphorylation of this complex was correlatedwith the adhesive capacity of the cadherins (Beherns et al., J. Cell.Biol. 120:757-766 (1993)), a result which is consistent with thehypothesis that the adhesion between cells mediated by the cadherinsmight be regulated by their tyrosine phosphorylation levels asdetermined by homotypic interactions between receptor PTP s such as κand μ.

The finding that PTPs κ and μ mediated homotypic adhesion, together withthe somewhat restricted tissue distribution of these PTPs (Jiang et al.,(1993) supra and Gebbink et al., (1991) supra), has suggested thatadditional members of this family of adhesive enzymes might exist. Herewe report the cloning and characterization of the third member of thisreceptor PTP family, termed PTP λ. The PTP λ polypeptide reported herecontains structural motifs that are very similar to those found in PTP κand μ. In addition, this novel PTP λ receptor reveals a tissuedistribution that is divergent from that previously described for theother members of this family.

SUMMARY OF THE INVENTION

We have analyzed a large number of PTPs from a primitive murinehematopoietic cell population using consensus PCR. From this populationwe have cloned a novel receptor protein tyrosine phosphorylasepolypeptide which is related to the receptor PTPs κ and μ. We havedesignated this novel protein tyrosine phosphorylase as the "PTP λ".Unlike other known receptor PTP polypeptides, PTP λ is predominantlyexpressed in mammalian adult brain, lung and kidney tissues butpredominantly lacks expression in mammalian adult liver tissue.

Accordingly, the present invention concerns an isolated receptor proteintyrosine phosphatase polypeptide (PTP ) λ, which

(1) is predominantly expressed in adult mammalian brain, lung and kidneytissue; and

(2) predominantly lacks expression in adult mammalian liver tissue,

wherein said polypeptide is capable of dephosphorylating phosphorylatedtyrosine residues.

The present invention also concerns derivatives of these novel PTPpolypeptides which substantially retain the ability to dephosphorylatephosphorylated tyrosine residues.

A preferred group of the PTP polypeptides of the present inventionincludes a polypeptide comprising the amino acid sequence shown in FIG.1 (SEQ ID NO:2); a further mammalian homologue of amino acid sequenceshown in FIG. 1 and a derivative of any of the above polypeptides whichsubstantially retain the ability to dephosphorylate tyrosine residues.

In another aspect, the present invention is directed to agonists andantagonists of the above novel PTP polypeptides.

In yet another aspect, the present invention concerns isolated nucleicacid molecules encoding the novel PTP polypeptides disclosed herein.

In a further aspect, the invention concerns vectors comprising nucleicacid encoding the novel PTP polypeptides herein, operably linked tocontrol sequences recognized by a host cell transformed with the vector,and to cells transformed with such vectors.

In a still further aspect of the present invention, there are providedantibodies capable of specific binding to the novel PTP polypeptides ofthis invention, and hybridoma cell lines producing such antibodies. Theantibodies may be agonist antibodies, which stimulate the ability of thenovel PTP polypeptides of the present invention to dephosphorylatetyrosines, or antagonist antibodies, which block this activity.

In yet a further aspect of the present invention, there is providedmethods for producing the PTP polypeptides of the present inventioncomprising transforming a host cell with nucleic acid encoding saidpolypeptide, culturing the transformed cell and recovering saidpolypeptide from the cell culture.

The present invention further concerns an assay for identifying anantagonist or an agonist of a novel PTP polypeptide of the presentinvention, which comprises contacting a phosphatase domain of the PTPpolypeptide with a candidate antagonist or agonist, and monitoring theability of the phosphatase domain to dephosphorylate tyrosine residues.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIGS. 1A-1I. The cDNA and derived protein sequence of PTP λ. Illustratedis the cDNA (SEQ ID NO:1) and derived protein sequence (SEQ ID NO:2) ofthe full length PTP λ clone homologous to a small PCR fragment derivedfrom hematopoietic progenitor cells using consensus PTP primers. Aminoacids are presented by their standard one-letter designations.

FIGS. 2A-2E. Homology between PTP λ, PTP κ and PTP μ. Illustrated asboxed residues are the amino acid homologies between the PTP λ(ptplambda) (SEQ ID NO:2), PTP κ (ptpkappa) (SEQ ID NO:3) and PTP μ(ptpmu) (SEQ ID NO:4) polypeptides. Amino acids are presented by theirstandard one-letter designations. Also shown above the amino acidsequences are the domains predicted previously from PTP κ and PTP μ.These domains include the signal sequence (SS), the MAM (mePrin, A5, PTPμ), immunoglobulin-like (IgG), fibronectin type III-like (FnIII),transmembrane domain (TMD), cadherin-like (Cadherin) and dualphosphatase domains (PTPase I and PTPase II).

FIG. 3. Comparative domain structures of PTP λ, PTP κ and PTP μ.Illustrated are the percent amino acid homologies between the variousdomains of the PTP λ, PTP κ and PTP μ polypeptides. These domainsinclude the signal sequence (SS), the MAM (mePrin, A5, PTP μ),immunoglobulin-like (IgG), fibronectin type III-like (FnIII),transmembrane domain (TMD), cadherin-like (Cadherin) and dualphosphatase domains (PTPase I and PTPase II).

FIG. 4. Tyrosine phosphatase activity of PTP λ immunoprecipitates fromPC 12 cells. Lysates of PC 12 cells were immunoprecipitated with eitherpreimmune antibody (Pre-immune) or antibody directed against thecytoplasmic domain of the PTP λ polypeptide (AntiPTP λ). Theimmunoprecipitates were incubated with two different immobilizedtyrosine phosphorylated peptides (PPS1 and PPS2) using a commerciallyavailable tyrosine phosphatase assay kit. Immunoprecipitates were doneeither in the absence or presence of the tyrosine phosphatase inhibitorvanadate. Tyrosine phosphatase activity was determined by examining theresidual binding of an anti-phosphotyrosine antibody to the immobilizedpeptide, so that a decreased OD₄₀₅ correlates with tyrosine phosphataseactivity.

FIG. 5. Northern blot analysis of PTP λ expression. Commerciallyavailable northern blots were probed with a ³² P-labeled fragment of PTPλ using standard hybridization conditions. The blot on the leftillustrates the PTP λ transcript in RNA obtained from murine embryos atthe developmental day shown in the figure. The blot on the rightillustrates an analysis of the PTP λ transcript in RNA from a. heart, b.brain, c. spleen, d. lung, e. liver, f. skeletal muscle, g. kidney andh. testis.

FIGS. 6A-6I. PTP λ mRNA Expression In the E15.5 Rat Embryo. Emulsionautoradiographs of a sagittal embryo section (A), and highermagnifications of embryonic midbrain (C), spinal cord (D), kidney (F),and lung hybridized with a ³³ P-UTP labeled PTP λ antisense probe areshown. Opposed to the darkfield autoradiographs are the correspondinglightfield images of the sagittal embryo section (B), kidney (G), andlung (I). Hybridization using a PTP λ sense strand control probe isshown in an E15.5 embryonic spinal cord section (E). (A,B,C,D,E) Bar,1.0 mm; (F,G,H,I) Bar, 0.2 mm.

FIGS. 7A-7F. PTP λ mRNA Expression in P1 and Adult Rat Brain. Emulsionautoradiographs of coronal sections of P1 rat brain (A,B,C) and adultrat brain (D,E) hybridized with a ³³ P-UTP labeled PTP λ antisense probeare shown. Coronal sections of the P1 brain are at the level of theseptum (A), hippocampus (B), and substantia nigra (C). For the adultanimal, coronal brain sections are at the level of the septum (D) andthe hippocampus and substantia nigra (E). Hybridization using a PTP λsense strand control probe is shown in an adult coronal section at thelevel of the substantia nigra (F). (A,B,C) Bar, 1.0 mm; (D,E,F) Bar, 1.0mm.

FIG. 8. Expression of PTP λ in PC 12 cells. Illustrated is the PTP λtranscript observed in RNA of PC 12 cells either untreated (-) ortreated (+) with 10 ng/ml of nerve growth factor (NGF) for the daysshown at the top of the figure. The lower blot shows the β-actin signalobtained for each of the RNAs.

FIGS. 9A-9D. Immunofluorescence analysis of PTP λ expression in PC 12cells. PC 12 cells were either left untreated or treated with 10 ng/mlnerve growth factor (NGF) for 7 days to induce neurite formation. At theend of this time, the cells were permeabilized and stained with eitherpre-immune serum or antibodies directed against the intracellular domainof PTP λ. Cells were washed and observed by confocal fluorescencemicroscopy. Panel A shows the results without NGF and with pre-immuneserum. Panel B shows the results without NGF and with anti-PTP λ serum.Panel C shows the results with NGF and anti-PTP λ serum. Panel D showsthe results obtained with NGF and anti-PTP λ serum at a highermagnification than in Panel C. The arrows show positively stainedextended neurites.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

The phrases "receptor protein tyrosine phosphatase λ", "protein tyrosinephosphatase λ" and "PTP λ" are used interchangeably and refer to anative membrane-bound protein tyrosine phosphatase polypeptide which (1)is predominantly expressed in adult mammalian brain, lung and kidneytissue and (2) predominantly lacks expression in adult mammalian livertissue, wherein the polypeptide is capable of dephosphorylatingphosphorylated tyrosine residues. The above terms are also intended toencompass functional derivatives of such native tyrosine phosphatases.

The term "native tyrosine phosphatase" in this context refers to anaturally occurring tyrosine phosphatase polypeptide, having thedescribed properties, of any human or non-human animal species, with orwithout the initiating methionine, whether purified from the nativesource, synthesized, produced by recombinant DNA technology or by anycombination of these and/or other methods. Native PTP λ specificallyincludes the native murine PTP λ protein shown in FIG. 1 (SEQ ID NO:2).

A "functional derivative" of a polypeptide is a compound having aqualitative biological activity in common with the native polypeptide.Thus, a functional derivative of a native PTP λ polypeptide is acompound that has a qualitative biological activity in common with anative PTP λ polypeptide, for example, as being capable ofdephosphorylating phosphorylated tyrosine residues. "Functionalderivatives" include, but are not limited to, fragments of nativepolypeptides from any animal species (including humans), derivatives ofnative (human and non-human) polypeptides and their fragments,glycosylation variants of a native polypeptide, and peptide andnon-peptide analogs of native polypeptides, provided that they have abiological activity in common with a respective native polypeptide."Fragments" comprise regions within the sequence of a mature nativepolypeptide. The term "derivative" is used to define amino acid sequencevariants, and covalent modifications of a native polypeptide."Non-peptide analogs" are organic compounds which display substantiallythe same surface as peptide analogs of the native polypeptides. Thus,the non-peptide analogs of the native PTP λ of the present invention areorganic compounds which display substantially the same surface aspeptide analogs of the native PTP λ. Such compounds interact with othermolecules in a similar fashion as the peptide analogs, and mimic abiological activity of a native PTP λ of the present invention. Thepolypeptide functional derivatives of the native PTP λ of the presentinvention preferably have at least about 65%, more preferably at leastabout 75%, even more preferably at least about 85%, most preferably atleast about 95% overall sequence homology with the amino acid sequenceshown in FIG. 1 (SEQ ID NO:2) and substantially retain the ability todephosphorylate phosphorylated tyrosine residues.

The term "biological activity" in the context of the definition offunctional derivatives is defined as the possession of at least oneadhesive, regulatory or effector function qualitatively in common with anative polypeptide (e.g. PTP λ). The functional derivatives of thenative PTP λ of the present invention are unified by their qualitativeability to dephosphorylate phosphorylated tyrosine residues. Preferably,the functional derivatives of the native PTP λ polypeptides of thepresent invention qualitatively retain at least one of the followingbiological properties of the native molecules: mediation of celladhesion, and involvement in neural pathfinding.

The terms "covalent modification" and "covalent derivatives" are usedinterchangeably and include, but are not limited to, modifications of anative polypeptide or a fragment thereof with an organic proteinaceousor non-proteinaceous derivatizing agent, fusions to heterologouspolypeptide sequences, and post-translational modifications. Covalentmodifications are traditionally introduced by reacting targeted aminoacid residues with an organic derivatizing agent that is capable ofreacting with selected sides or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. Certain post-translational modifications are theresult of the action of recombinant host cells on the expressedpolypeptide. Glutaminyl and asparaginyl residues are frequentlypost-translationally deamidated to the corresponding glutamyl andaspartyl residues. Alternatively, these residues are deamidated undermildly acidic conditions. Other post-translational modifications includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl, tyrosine or threonyl residues, methylation of the a-aminogroups of lysine, arginine, and histidine side chains [T. E. Creighton,Proteins: Structure and Molecular Properties, W.H. Freeman & Co., SanFrancisco, pp. 79-86 (1983)]. Covalent derivatives/modificationsspecifically include fusion proteins comprising native PTP λ sequencesof the present invention and their amino acid sequence variants, such asimmunoadhesins, and N-terminal fusions to heterologous signal sequences.

"Predominantly expressed", "predominant expression" and grammaticalequivalents thereof is defined as a level of expression of a nucleicacid encoding an amino acid sequence which is easily detectable usingnorthern blot analysis under stringent conditions.

"Identity" or "homology" with respect to a native polypeptide and itsfunctional derivative is defined herein as the percentage of amino acidresidues in the candidate sequence that are identical with the residuesof a corresponding native polypeptide, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and not considering any conservative substitutions as part of thesequence identity. Neither N- or C-terminal extensions nor insertionsshall be construed as reducing identity or homology. Methods andcomputer programs for the alignment are well known in the art.

The term "agonist" is used to refer to peptide and non-peptide analogsof the native PTP λ of the present invention and to antibodiesspecifically binding native PTP λ provided that they retain at least onebiological activity of a native PTP λ. Preferably, the agonists of thepresent invention retain the qualitative ability to dephosphorylatephosphorylated tyrosine residues.

The term "antagonist" is used to refer to a molecule inhibiting abiological activity of a native PTP λ of the present invention.Preferably, the antagonists herein inhibit the ability of the PTP λ ofthe present invention to dephosphorylate tyrosines. Preferredantagonists essentially completely block tyrosine dephosphorylationcaused by PTP λ.

Ordinarily, the terms "amino acid" and "amino acids" refer to allnaturally occurring L-α-amino acids. In some embodiments, however,D-amino acids may be present in the polypeptides or peptides of thepresent invention in order to facilitate conformational restriction. Forexample, in order to facilitate disulfide bond formation and stability,a D amino acid cysteine may be provided at one or both termini of apeptide functional derivative or peptide antagonist of the native PTP λof the present invention. The amino acids are identified by either thesingle-letter or three-letter designations:

    ______________________________________                                        Asp   D      aspartic acid                                                                              Ile   I    isoleucine                                 Thr T threonine Leu L leucine                                                 Ser S serine Tyr Y tyrosine                                                   Glu E glutamic acid Phe F phenylalanine                                       Pro P proline His H histidine                                                 Gly G glycine Lys K lysine                                                    Ala A alanine Arg R arginine                                                  Cys C cysteine Trp W tryptophan                                               Val V valine Gln Q glutamine                                                  Met M methionine Asn N asparagine                                           ______________________________________                                    

The amino acids may be classified according to the chemical compositionand properties of their side chains. They are broadly classified intotwo groups, charged and uncharged. Each of these groups is divided intosubgroups to classify the amino acids more accurately:

I. Charged Amino Acids

Acidic Residues: aspartic acid, glutamic acid

Basic Residues: lysine, arginine, histidine

II. Uncharged Amino Acids

Hydrophilic Residues: serine, threonine, asparagine, glutamine

Aliphatic Residues: glycine, alanine, valine, leucine, isoleucine

Non-polar Residues: cysteine, methionine, proline

Aromatic Residues: phenylalanine, tyrosine, tryptophan

The term "amino acid sequence variant" refers to molecules with somedifferences in their amino acid sequences as compared to a native aminoacid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative sequence. Immediately adjacent to an amino acid means connectedto either the α-carboxy or α-amino functional group of the amino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

"Antibodies (Abs)" and "immunoglobulins (Igs)" are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains (Clothia et al., J.Mol. Biol., 186, 651-663 [1985]; Novotny and Haber, Proc. Natl. Acad.Sci. USA 82, 4592-4596 [1985]).

The term "variable" refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthrough the variable domains of antibodies. It is concentrated in threesegments called complementarity determining regions (CDRs) orhypervariable regions both in the light chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen binding site of antibodies (see Kabat, E. A. et al., Sequencesof Proteins of Immunological Interest, National Institute of Health,Bethesda, Md. [1991]). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual "Fc" fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab')₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

"Fv" is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H) -V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab' fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab'-SH is the designationherein for Fab' in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab')₂ antibody fragments originally wereproduced as pairs of Fab' fragments which have hinge cysteines betweenthem. Other, chemical couplings of antibody fragments are also known.

The light chains of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called a, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The term "antibody" is used in the broadest sense and specificallycovers single monoclonal antibodies (including agonist and antagonistantibodies), antibody compositions with polyepitopic specificity, aswell as antibody fragments (e.g., Fab, F(ab')₂, and Fv), so long as theyexhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier "monoclonal"indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler & Milstein, Nature 2:495 (1975), or may be made byrecombinant DNA methods [see, e.g. U.S. Pat. No. 4,816,567 (Cabilly etal.)].

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567(Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA 81,6851-6855 [1984]).

"Humanized" forms of non-human (e.g. murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibody maycomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin. For further details see:Jones et al., Nature 321, 522-525 [1986]; Reichmann et al., Nature 332,323-329 [1988]; EP-B-239 400 published Sep. 30, 1987; Presta, Curr. Op.Struct. Biol. 2 593-596 [1992]; and EP-B-451 216 published Jan. 24,1996).

In the context of the present invention the expressions "cell", "cellline", and "cell culture" are used interchangeably, and all suchdesignations include progeny. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological property, as screened for in the originally transformed cell,are included.

The terms "replicable expression vector" and "expression vector" referto a piece of DNA, usually double-stranded, which may have inserted intoit a piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

The term "control sequences" refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

"Immunoadhesins" or "PTP λ-immunoglobulin chimeras" are chimericantibody-like molecules that combine the functional domain(s) of abinding protein (usually a receptor, a cell-adhesion molecule or aligand) with the an immunoglobulin sequence. The most common example ofthis type of fusion protein combines the hinge and Fc regions of animmunoglobulin (Ig) with domains of a cell-surface receptor thatrecognizes a specific ligand. This type of molecule is called an"immunoadhesin", because it combines "immune" and "adhesion" functions;other frequently used names are "Ig-chimera", "Ig-" or "Fc-fusionprotein", or "receptor-globulin."

"Oligonucleotides" are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods[such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as those described in EP 266,032, publishedMay 4, 1988, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., Nucl. Acids Res. 14, 5399 (1986). They arethen purified on polyacrylamide gels.

Hybridization is preferably performed under "stringent conditions" whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C., or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1%SDS. Yet another example is hybridization using a buffer of 10% dextransulfate, 2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegration. Depending on the host cell used, transformation is doneusing standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride, as described by Cohen, S. N.,Proc. Natl. Acad. Sci. (USA), 69, 2110 (1972) and Mandel et al. J. Mol.Biol. 53, 154 (1970), is generally used for prokaryotes or other cellsthat contain substantial cell-wall barriers. For mammalian cells withoutsuch cell walls, the calcium phosphate precipitation method of Graham,F. and van der Eb, A., Virology, 52, 456-457 (1978) is preferred.General aspects of mammalian cell host system transformations have beendescribed by Axel in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformations into yeast are typically carried out according to themethod of Van Solingen, P., et al. J. Bact., 130, 946 (1977) and Hsiao,C. L., et al. Proc. Natl. Acad. Sci. (USA) 76, 3829 (1979). However,other methods for introducing DNA into cells such as by nuclearinjection, electroporation or by protoplast fusion may also be used.

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide or agarose gelby electrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., Nucleic Acids Res. 9,6103-6114 (1981) and D. Goeddel et al., Nucleic Acids Res. 8, 4057(1980).

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al. 1982,supra, p. 146). Unless otherwise provided, ligation may be accomplishedusing known buffers and conditions with 10 units of T4 DNA ligase("ligase") per 0.5 mg of approximately equimolar amounts of the DNAfragments to be ligated.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofManiatis et al. 1982, supra, p. 90, may be used.

B. Production of PTP λ by recombinant DNA technology

1. Identification and isolation of nucleic acid encoding PTP λ

The native PTP λ proteins of the present invention may be isolated fromcDNA or genomic libraries. For example, a suitable cDNA library can beconstructed by obtaining polyadenylated mRNA from cells known to expressthe desired PTP λ protein, and using the mRNA as a template tosynthesize double stranded cDNA. Suitable sources of the mRNA are murineprimitive hematopoietic cells and PC 12 cells. mRNA encoding the nativePTP λ of the present invention is expressed, for example, in tissuesderived from adult brain, lung, kidney, heart, skeletal muscle andtestis. The gene encoding the novel PTP λ polypeptide of the presentinvention can also be obtained from a genomic library, such as a humangenomic cosmid library, or a mouse-derived embryonic cell (ES) genomiclibrary.

Libraries, either cDNA or genomic, are then screened with probesdesigned to identify the gene of interest or the protein encoded by it.For cDNA expression libraries, suitable probes include monoclonal andpolyclonal antibodies that recognize and specifically bind to a PTP λpolypeptide. For cDNA libraries, suitable probes include carefullyselected oligonucleotide probes (usually of about 20-80 bases in length)that encode known or suspected portions of a PTP λ polypeptide from thesame or different species, and/or complementary or homologous cDNAs orfragments thereof that encode the same or a similar gene. Appropriateprobes for screening genomic DNA libraries include, without limitation,oligonucleotides, cDNAs, or fragments thereof that encode the same or asimilar gene, and/or homologous genomic DNAs or fragments thereof.Screening the cDNA or genomic library with the selected probe may beconducted using standard procedures as described in Chapters 10-12 ofSambrook et al., Molecular Cloning: A Laboratory Manual, New York, ColdSpring Harbor Laboratory Press, 1989.

If DNA encoding an enzyme of the present invention is isolated by usingcarefully selected oligonucleotide sequences to screen cDNA librariesfrom various tissues, the oligonucleotide sequences selected as probesshould be sufficient in length and sufficiently unambiguous that falsepositives are minimized. The actual nucleotide sequence(s) is/areusually designed based on regions which have the least codon redundance.The oligonucleotides may be degenerate at one or more positions. The useof degenerate oligonucleotides is of particular importance where alibrary is screened from a species in which preferential codon usage isnot known.

The oligonucleotide must be labeled such that it can be detected uponhybridization to DNA in the library being screened. The preferred methodof labeling is to use ATP (e.g., γ³² P) and polynucleotide kinase toradiolabel the 5' end of the oligonucleotide. However, other methods maybe used to label the oligonucleotide, including, but not limited to,biotinylation or enzyme labeling.

cDNAs encoding PTP λ can also be identified and isolated by other knowntechniques of recombinant DNA technology, such as by direct expressioncloning, or by using the polymerase chain reaction (PCR) as described inU.S. Pat. No. 4,683,195, issued Jul. 28, 1987, in section 14 of Sambrooket al., supra, or in Chapter 15 of Current Protocols in MolecularBiology, Ausubel et al. eds., Greene Publishing Associates andWiley-Interscience 1991. The use of the PCR technique for obtaining cDNAencoding murine PTP λ is also illustrated in the examples.

Once cDNA encoding a PTP λ enzyme from one species has been isolated,cDNAs from other species can also be obtained by cross-specieshybridization. According to this approach, human or other mammalian cDNAor genomic libraries are probed by labeled oligonucleotide sequencesselected from known PTP λ sequences (such as murine PTP λ) in accordwith known criteria, among which is that the sequence should besufficient in length and sufficiently unambiguous that false positivesare minimized. Typically, a ³² P-labeled oligonucleotide having about 30to 50 bases is sufficient, particularly if the oligonucleotide containsone or more codons for methionine or tryptophan. Isolated nucleic acidwill be DNA that is identified and separated from contaminant nucleicacid encoding other polypeptides from the source of nucleic acid.Hybridization is preferably performed under "stringent conditions", asherein above defined.

Once the sequence is known, the gene encoding a particular PTP λpolypeptide can also be obtained by chemical synthesis, following one ofthe methods described in Engels and Uhlmann, Agnew. Chem. Int. Ed. Engl.28, 716 (1989). These methods include triester, phosphite,phosphoramidite and H-phosphonate methods, PCR and other autoprimermethods, and oligonucleotide syntheses on solid supports.

2. Cloning and expression of nucleic acid encoding PTP λ

Once the nucleic acid encoding PTP λ is available, it is generallyligated into a replicable expression vector for further cloning(amplification of the DNA), or for expression.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA ofexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of the above listed components, the desired coding and controlsequences, employs standard ligation techniques. Isolated plasmids orDNA fragments are cleaved, tailored, and religated in the form desiredto generate the plasmids required. For analysis to confirm correctsequences in plasmids constructed, the ligation mixtures are commonlyused to transform E. coli cells, e.g. E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

The polypeptides of the present invention may be expressed in a varietyof prokaryotic and eukaryotic host cells. Suitable prokaryotes includegram negative or gram positive organisms, for example E. coli orbacilli. A preferred cloning host is E. coli 294 (ATCC 31,446) althoughother gram negative or gram positive prokaryotes such as E. coli B, E.coli X1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), Pseudomonasspecies, or Serratia Marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors herein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species and strains are commonly available and useful herein, such as S.pombe [Beach and Nurse, Nature 290, 140 (1981)], Kluyveromyces lactis[Louvencourt et al., J. Bacteriol. 737 (1983)]; yarrowia (EP 402,226);Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234),Neurospora crassa [Case et al., Proc. Natl. Acad. Sci. USA 76, 5259-5263(1979)]; and Aspergillus hosts such as A. nidulans [Ballance et al.,Biochem. Biophys. Res. Commun. 112, 284-289 (1983); Tilburn et al., Gene26, 205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA 81,1470-1474 (1984)] and A. niger [Kelly and Hynes, EMBO J. 4, 475-479(1985)].

Suitable host cells may also derive from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al., Nature 315, 592-594 (1985). Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa californica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the PTP λ DNA. During incubation of the plant cell culture withA. tumefaciens, the DNA encoding a PTP λ polypeptide is transferred tothe plant cell host such that it is transfected, and will, underappropriate conditions, express the PTP λ DNA. In addition, regulatoryand signal sequences compatible with plant cells are available, such asthe nopaline synthase promoter and polyadenylation signal sequences.Depicker et al., J. Mol. Appl. Gen. 1, 561 (1982). In addition, DNAsegments isolated from the upstream region of the T-DNA 780 gene arecapable of activating or increasing transcription levels ofplant-expressible genes in recombinant DNA-containing plant tissue. SeeEP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) is per se well known.See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973).Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cellline [293 or 293 cells subcloned for growth in suspension culture,Graham et al., J. Gen. Virol. 36, 59 (1977)]; baby hamster kidney cells9BHK, ATCC CCL 10); Chinese hamster ovary cells/DHFR [CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA 77, 4216 (1980)]; mouse sertollicells [TM4, Mather, Biol. Reprod. 23, 243-251 (1980)]; monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells [Mather et al., Annals N.Y. Acad. Sci. 383, 44068 (1982)]; MRC5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). Preferredhost cells are human embryonic kidney 293 and Chinese hamster ovarycells.

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding a PTP λ polypeptide. In general, transient expressioninvolves the use of an expression vector that is able to replicateefficiently in a host cell, such that the host cell accumulates manycopies of the expression vector and, in turn, synthesizes high levels ofa desired polypeptide encoded by the expression vector. Transientsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded byclones DNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of a PTP λ polypeptide.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the PTP λ polypeptides in recombinant vertebrate cellculture are described in Getting et al., Nature 293, 620-625 (1981);Mantel et al., Nature 281, 40-46 (1979); Levinson et al.; EP 117,060 andEP 117,058. Particularly useful plasmids for mammalian cell cultureexpression of the PTP λ polypeptides are pRK5 (EP 307,247) or pSVI6B(PCT Publication No. WO 91/08291).

Other cloning and expression vectors suitable for the expression of thePTP λ polypeptides of the present invention in a variety of host cellsare, for example, described in EP 457,758 published Nov. 27, 1991. Alarge variety of expression vectors are now commercially available. Anexemplary commercial yeast expression vector is pPIC.9 (Invitrogen),while an commercially available expression vector suitable fortransformation of E. coli cells is PET15b (Novagen).

C. Culturing the host cells

Prokaryote cells used to produced the PTP λ polypeptides of thisinvention are cultured in suitable media as describe generally inSambrook et al., supra.

Mammalian cells can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium(MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium(DMEM, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enzymol. 58, 44(1979); Barnes and Sato, Anal. Biochem. 102, 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195or U.S. Pat. No. Re. 30,985 may be used as culture media for the hostcells. Any of these media may be supplemented as necessary with hormonesand/or other growth factors (such as insulin, transferrin, or epidermalgrowth factor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

The host cells referred to in this disclosure encompass cells in invitro cell culture as well as cells that are within a host animal orplant.

It is further envisioned that the PTP λ polypeptides of this inventionmay be produced by homologous recombination, or with recombinantproduction methods utilizing control elements introduced into cellsalready containing DNA encoding the particular PTP λ polypeptide.

D. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA 77, 5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³² P. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hse et al., Am. J. Clin. Pharm.75, 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native PTP λ polypeptide, or against a synthetic peptide basedon the DNA sequence provided herein as described further hereinbelow.

E. Amino Acid Sequence Variants of Native PTP λ Polypeptides

Amino acid sequence variants of native PTP λ polypeptides are preparedby methods known in the art by introducing appropriate nucleotidechanges into a PTP λ DNA, or by in vitro synthesis of the desiredpolypeptide. There are two principal variables in the construction ofamino acid sequence variants: the location of the mutation site and thenature of the mutation. With the exception of naturally-occurringalleles, which do not require the manipulation of the DNA sequenceencoding the PTP λ polypeptide, the amino acid sequence variants of PTPλ polypeptides are preferably constructed by mutating the DNA, either toarrive at an allele or an amino acid sequence variant that does notoccur in nature.

One group of the mutations will be created within at least one of thephosphatase domains (PTPaseI and/or PTPaseII) of a native PTP λ protein.In view of the involvement of these domains in the enzymatic activity ofPTP λ, amino acid alterations within these domains are expected toresult in marked changes in the enzymatic properties of the nativeproteins. Non-conservative substitutions might ultimately result in PTPλ variants which lose the ability to dephosphatase tyrosines and will,therefore, be useful as antagonists of native PTP λ. PTP λ variantsmutated to enhance the enzymatic activity of the native proteins mayalso be obtained, and will find use, for example, as potent mediators ofcell adhesion.

Similarly, amino acid alterations in the MAM of IgG domains of thenative PTP λ proteins are expected to affect the ability of thesereceptors to mediate homotypic cell adhesion, and the specificity of thehomophilic interaction mediated.

Alternatively or in addition, amino acid alterations can be made atsites that differ in PTP λ proteins from various species, or in highlyconserved regions, depending on the goal to be achieved. Sites at suchlocations will typically be modified in series, e.g. by (1) substitutingfirst with conservative choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue orresidues, or (3) inserting residues of the same or different classadjacent to the located site, or combinations of options 1-3. Onehelpful technique is called "alanine scanning" (Cunningham and Wells,Science 244, 1081-1085 [1989]). The replacement of sequence motifswithin the MAM, IgG, FNIII or PTPase domains of the native PTP λproteins of the present invention by sequences from native PTP κ and/orPTP μ receptors is expected to result in variants having alteredspecificities.

In yet another group of the variant PTP λ polypeptides of the presentinvention, one or more of the functionally less significant domains maybe deleted or inactivated. For example, the deletion or inactivation ofthe transmembrane domain yields soluble variants of the native protein.Alternatively, or in addition, the cytoplasmic domain may be deleted,truncated or otherwise altered.

Naturally-occurring amino acids are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophobic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Substantial changes in function or immunological identityare made by selecting substitutions that are less conservative, i.e.differ more significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of substitution, forexample as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the properties of the novel native PTP λpolypeptides of the present invention will be those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g. lysyl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, e.g., glutamyl or aspartyl; or (d) a residue having a bulkyside chain, e.g., phenylalanine, is substituted for (or by) one nothaving a side chain, e.g. glycine.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Typically, the transmembrane and cytoplasmic domains, oronly the cytoplasmic domains are deleted. However, deletion from theC-terminal to any other suitable N-terminal to the transmembrane regionwhich preserves the biological activity or immunologicalcross-reactivity of a native PTP λ is suitable.

A preferred class of substitutional and/or deletional variants of thepresent invention are those involving a transmembrane region of a novelPTP λ molecule. Transmembrane regions are highly hydrophobic orlipophilic domains that are the proper size to span the lipid bilayer ofthe cellular membrane. They are believed to anchor the PTP λ protein inthe cell membrane and allow for homotypic complex formation.Inactivation of the transmembrane domain, typically by deletion orsubstitution of transmembrane domain hydroxylation residues, willfacilitate recovery and formulation by reducing its cellular or membranelipid affinity and improving its aqueous solubility. If thetransmembrane and cytoplasmic domains are deleted one avoids theintroduction of potentially immunogenic epitopes, whether by exposure ofotherwise intracellular polypeptides that might be recognized by thebody as foreign or by insertion of heterologous polypeptides that arepotentially immunogenic. Inactivation of the membrane binding functionis accomplished by deletion of sufficient residues to produce asubstantially hydrophilic hydropathy profile at this site or bysubstituting with heterologous residues which accomplish the sameresult.

A principle advantage of the transmembrane inactivated variants of thePTP λ polypeptides of the present invention is that they may be secretedinto the culture medium of recombinant hosts. These variants are solublein body fluids such as blood and do not have an appreciable affinity forcell membrane lipids, thus considerably simplifying their recovery fromrecombinant cell culture. As a general proposition, such solublevariants will not have a functional transmembrane domain and preferablywill not have a functional cytoplasmic domain. For example, thetransmembrane domain may be substituted by any amino acid sequence, e.g.a random or predetermined sequences of about 5 to 50 serine, threonine,lysine, arginine, glutamine, aspartic acid and like hydrophilicresidues, which altogether exhibit a hydrophilic hydropathy profile.Like the deletional (truncated) soluble variants, these variants aresecreted into the culture medium of recombinant hosts.

Amino acid insertions include amino- and/or carboxyl-terminal fusionsranging in length from one residue to polypeptides containing a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Intrasequence insertions (i.e. insertionswithin the PTP λ protein amino acid sequence) may range generally fromabout 1 to 10 residues, more preferably 1 to 5 residues, more preferably1 to 3 residues. Examples of terminal insertions include PTP λpolypeptides with an N-terminal methionyl residue, an artifact of itsdirect expression in bacterial recombinant cell culture, and fusion of aheterologous N-terminal signal sequence to the N-terminus of the PTP λmolecule to facilitate the secretion of the mature PTP λ fromrecombinant host cells. Such signal sequences will generally be obtainedfrom, and thus homologous to, the intended host cell species. Suitablesequences include STII or Ipp for E. coli, alpha factor for yeast, andviral signals such as herpes gD for mammalian cells.

Other insertional variants of the native PTP λ molecules include thefusion of the N- or C-terminus of the PTP λ molecule to immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions),albumin, or ferritin, as described in WO 89/02922 published on Apr. 6,1989.

Further insertional variants are immunologically active derivatives ofthe novel PTP λ polypeptides, which comprise the PTP polypeptide and apolypeptide containing an epitope of an immunologically competentextraneous polypeptide, i.e. a polypeptide which is capable of elicitingan immune response in the animal to which the fusion is to beadministered or which is capable of being bound by an antibody raisedagainst an extraneous polypeptide. Typical examples of suchimmunologically competent polypeptides are allergens, autoimmuneepitopes, or other potent immunogens or antigens recognized bypre-existing antibodies in the fusion recipient, including bacterialpolypeptides such as trpLE, β-galactosidase, viral polypeptides such asherpes gD protein, and the like.

Immunogenic fusions are produced by cross-linking in vitro or byrecombinant cell culture transformed with DNA encoding an immunogenicpolypeptide. It is preferable that the immunogenic fusion be one inwhich the immunogenic sequence is joined to or inserted into a novel PTPλ molecule or fragment thereof by (a) peptide bond(s). These productstherefore consist of a linear polypeptide chain containing the PTP λepitope and at least one epitope foreign to the PTP λ polypeptide. Itwill be understood that it is within the scope of this invention tointroduce the epitopes anywhere within a PTP λ molecule of the presentinvention or a fragment thereof. These immunogenic insertions areparticularly useful when formulated into a pharmacologically acceptablecarrier and administered to a subject in order to raise antibodiesagainst the PTP λ molecule, which antibodies in turn are useful asdiagnostics, in tissue-typing, or in purification of the novel PTP λpolypeptides by immunoaffinity techniques known per se. Alternatively,in the purification of the PTP λ polypeptides of the present invention,binding partners for the fused extraneous polypeptide, e.g. antibodies,receptors or ligands, are used to adsorb the fusion from impureadmixtures, after which the fusion is eluted and, if desired, the novelPTP λ is recovered from the fusion, e.g. by enzymatic cleavage.

Since it is often difficult to predict in advance the characteristics ofa variant PTP λ polypeptide, it will be appreciated that some screeningwill be needed to select the optimum variant.

After identifying the desired mutation(s), the gene encoding a PTP λvariant can, for example, be obtained by chemical synthesis ashereinabove described. More preferably, DNA encoding a PTP λ amino acidsequence variant is prepared by site-directed mutagenesis of DNA thatencodes an earlier prepared variant or a nonvariant version of the PTPλ. Site-directed (site-specific) mutagenesis allows the production ofPTP λ variants through the use of specific oligonucleotide sequencesthat encode the DNA sequence of the desired mutation, as well as asufficient number of adjacent nucleotides, to provide a primer sequenceof sufficient size and sequence complexity to form a stable duplex onboth sides of the deletion junction being traversed. Typically, a primerof about 20 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered. Ingeneral, the techniques of site-specific mutagenesis are well known inthe art, as exemplified by publications such as, Edelman et al., DNA 2,183 (1983). As will be appreciated, the site-specific mutagenesistechnique typically employs a phage vector that exists in both asingle-stranded and double-stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage, forexample, as disclosed by Messing et al., Third Cleveland Symposium onMacromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam(1981). This and other phage vectors are commercially available andtheir use is well known to those skilled in the art. A versatile andefficient procedure for the construction of oligodeoxyribonucleotidedirected site-specific mutations in DNA fragments using M13-derivedvectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res.10, 6487--6500 [1982]). Also, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153, 3 [1987]) may be employed to obtain single-stranded DNA.Alternatively, nucleotide substitutions are introduced by synthesizingthe appropriate DNA fragment in vitro, and amplifying it by PCRprocedures known in the art.

The PCR technique may also be used in creating amino acid sequencevariants of a PTP λ polypeptide. In a specific example of PCRmutagenesis, template plasmid DNA (1 μg) is linearized by digestion witha restriction endonuclease that has a unique recognition site in theplasmid DNA outside of the region to be amplified. Of this material, 100ng is added to a PCR mixture containing PCR buffer, which contains thefour deoxynucleotide triphosphates and is included in the GeneAmp® kits(obtained from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville,Calif.), and 25 pmole of each oligonucleotide primer, to a final volumeof 50 μl. The reaction mixture is overlayered with 35 μl mineral oil.The reaction is denatured for 5 minutes at 100° C., placed briefly onice, and then 1 μl Thermus aquaticus (Taq) DNA polymerase (5 units/ul),purchased from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville,Calif.) is added below the mineral oil layer. The reaction mixture isthen inserted into a DNA Thermal Cycler (purchased from Perkin-ElmerCetus) programmed as follows:

2 min. 55° C.,

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.,

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. [Gene 3, 315 (1985)].

Additionally, the so-called phagemid display method may be useful inmaking amino acid sequence variants of native or variant PTP λpolypeptides or their fragments. This method involves (a) constructing areplicable expression vector comprising a first gene encoding a receptorto be mutated, a second gene encoding at least a portion of a natural orwild-type phage coat protein wherein the first and second genes areheterologous, and a transcription regulatory element operably linked tothe first and second genes, thereby forming a gene fusion encoding afusion protein; (b) mutating the vector at one or more selectedpositions within the first gene thereby forming a family of relatedplasmids; (c) transforming suitable host cells with the plasmids; (d)infecting the transformed host cells with a helper phage having a geneencoding the phage coat protein; (e) culturing the transformed infectedhost cells under conditions suitable for forming recombinant phagemidparticles containing at least a portion of the plasmid and capable oftransforming the host, the conditions adjusted so that no more than aminor amount of phagemid particles display more than one copy of thefusion protein on the surface of the particle; (f) contacting thephagemid particles with a suitable antigen so that at least a portion ofthe phagemid particles bind to the antigen; and (g) separating thephagemid particles that bind from those that do not. Steps (d) through(g) can be repeated one or more times. Preferably in this method theplasmid is under tight control of the transcription regulatory element,and the culturing conditions are adjusted so that the amount or numberof phagemid particles displaying more than one copy of the fusionprotein on the surface of the particle is less than about 1%. Also,preferably, the amount of phagemid particles displaying more than onecopy of the fusion protein is less than 10% of the amount of phagemidparticles displaying a single copy of the fusion protein. Mostpreferably, the amount is less than 20%. Typically in this method, theexpression vector will further contain a secretory signal sequence fusedto the DNA encoding each subunit of the polypeptide and thetranscription regulatory element will be a promoter system. Preferredpromoter systems are selected from lac Z, λ_(PL), tac, T7 polymerase,tryptophan, and alkaline phosphatase promoters and combinations thereof.Also, normally the method will employ a helper phage selected fromM13K07, M13R408, M13-VCS, and Phi X 174. The preferred helper phage isM13K07, and the preferred coat protein is the M13 Phage gene III coatprotein. The preferred host is E. coli, and protease-deficient strainsof E. coli.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,supra, and Current Protocols in Molecular Biology, Ausubel et al. eds.,supra.

F. Glycosylation Variants

Glycosylation variants are included within the scope of the presentinvention. They include variants completely lacking in glycosylation(unglycosylated), variants having at least one less glycosylated sitethan the native form (deglycosylated) as well as variants in which theglycosylation has been changed. Included are deglycosylated andunglycosylated amino acid sequences variants, deglycosylated andunglycosylated native PTP λ, and other glycosylation variants. Forexample, substitutional or deletional mutagenesis may be employed toeliminate the N- or O-linked glycosylation sites in the a native orvariant PTP λ molecule of the present invention, e.g. the asparagineresidue may be deleted or substituted for another basic residue such aslysine or histidine. Alternatively, flanking residues making up theglycosylation site may be substituted or deleted, even though theasparagine residues remain unchanged, in order to prevent glycosylationby eliminating the glycosylation recognition site.

Additionally, unglycosylated PTP λ polypeptides which have theglycosylation sites of a native molecule may be produced in recombinantprokaryotic cell culture because prokaryotes are incapable ofintroducing glycosylation into polypeptides.

Glycosylation variants may be produced by selecting appropriate hostcells or by in vitro methods. Yeast and insect cells, for example,introduce glycosylation which varies significantly from that ofmammalian systems. Similarly, mammalian cells having a different species(e.g. hamster, murine, porcine, bovine or ovine), orliver, lymphin (e.g.lung, liver, lymphoid, mesenchymal or epidermal) than the source of thePTP λ polypeptide are routinely screened for the ability to introducevariant glycosylation as characterized for example by elevated levels ofmannose or variant ratios of mannose, fucose, sialic acid, and othersugars typically found in mammalian glycoproteins. In vitro processingof the PTP λ typically is accomplished by enzymatic hydrolysis, e.g.neuraminidase digestion.

G. Covalent Modification of PTP λ Polypeptides

Covalent modifications of PTP λ polypeptides are included within thescope herein. Such modifications are traditionally introduced byreacting targeted amino acid residues of the PTP λ polypeptides with anorganic derivatizing agent that is capable of reacting with selectedsides or terminal residues, or by harnessing mechanisms ofpost-translational modifications that function in selected recombinanthost cells. The resultant covalent derivatives are useful in programsdirected at identifying residues important for biological activity, forimmunoassays of the PTP λ polypeptide, or for the preparation ofanti-PTP λ antibodies for immunoaffinity purification of therecombinant. For example, complete inactivation of the biologicalactivity of the protein after reaction with ninhydrin would suggest thatat least one arginyl or lysyl residue is critical for its activity,whereafter the individual residues which were modified under theconditions selected are identified by isolation of a peptide fragmentcontaining the modified amino acid residue. Such modifications arewithin the ordinary skill in the art and are performed without undueexperimentation.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R') such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86[1983]), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of PTP λ polypeptides with polypeptides aswell as for cross-linking the PTP λ polypeptide to a water insolublesupport matrix or surface for use in assays or affinity purification. Inaddition, a study of interchain cross-links will provide directinformation on conformational structure. Commonly used cross-linkingagents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the α-amino groups of lysine, arginine,and histidine side chains [T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)].

Further derivatives of the PTP λ polypeptides herein are the so-called"immunoadhesins", which are chimeric antibody-like molecules combiningthe functional domain(s) of a binding protein (usually a receptor, acell-adhesion molecule or a ligand) with the an immunoglobulin sequence.The most common example of this type of fusion protein combines thehinge and Fc regions of an immunoglobulin (Ig) with domains of acell-surface receptor that recognizes a specific ligand. This type ofmolecule is called an "immunoadhesin", because it combines "immune" and"adhesion" functions; other frequently used names are "Ig-chimera","Ig-" or "Fc-fusion protein", or "receptor-globulin."

To date, more than fifty immunoadhesins have been reported in the art.lmmunoadhesins reported in the literature include, for example, fusionsof the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84,2936-2940 [1987]); CD4 (Capon et al., Nature 337, 525-531 [1989];Traunecker et al., Nature 339, 68-70 [1989]; Zettmeissl et al., DNA CellBiol. USA 9, 347-353 [1990]; Byrn et al., Nature 334, 667-670 [1990]);L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110,2221-2229 [1990]; Watson et al., Nature 349, 164-167 [1991]); E-selectin[Mulligan et al., J. Immunol. 151, 6410-17 [1993]; Jacob et al.,Biochemistry 34, 1210-1217 [1995]); P-selectin (Mulligan et al., supra;Hollenbaugh et al., Biochemistry 34, 5678-84 [1995]); ICAM-1 (Stauton etal., J. Exp. Med. 176, 1471-1476 [1992]; Martin et al., J. Virol. 67,3561-68 [1993]; Roep et al., Lancet 343, 1590-93 [1994]); ICAM-2 (Damleet al., J. Immunol. 148, 665-71 [1992]); ICAM-3 (Holness et al., J.Biol. Chem. 270, 877-84 [1995]); LFA-3 (Kanner et al., J. Immunol. 148,2-23-29 [1992]); L1 glycoprotein (Doherty et al., Neuron 14, 57-66[1995]); TNF-R1 (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88,10535-539 [1991]; Lesslauer et al., Eur. J. Immunol. 21, 2883-86 [1991];Peppel et al., J. Exp. Med. 174, 1483-1489 [1991]); TNF-R2 (Zack et al.,Proc. Natl. Acad. Sci. USA 90, 2335-39 [1993]; Wooley et al., J.Immunol. 151, 6602-07 [1993]); CD44 [Aruffo et al., Cell 61, 1303-1313(1990)]; CD28 and B7 [Linsley et al., J. Exp. Med. 173, 721-730 (1991)];CTLA-4 [Lisley et al., J. Exp. Med. 174, 561-569 (1991)]; CD22[Stamenkovic et al., Cell 66. 1133-1144 (1991)]; NP receptors [Bennettet al., J. Biol. Chem. 266, 23060-23067 (1991)]; IgE receptor α [Ridgwayand Gorman, J. Cell. Biol. 115, abstr. 1448 (1991)]; HGF receptor [Mark,M. R. et al., 1992, J. Biol. Chem. submitted]; IFN-γR α- and β-chain[Marsters et al., Proc. Natl. Acad. Sci. USA 92, 5401-05 [1995]); trk-A,-B, and -C (Shelton et al., J. Neurosci. 15, 477-91 [1995]); IL-2(Landolfi, J. Immunol. 146, 915-19 [1991]); IL-10 (Zheng et al., J.Immunol. 154, 5590-5600 [1995]).

The simplest and most straightforward immunoadhesin design combines thebinding region(s) of the `adhesin` protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparing the PTPλ-immunoglobulin chimeras of the present invention, nucleic acidencoding the desired PTP λ polypeptide will be fused C-terminally tonucleic acid encoding the N-terminus of an immunoglobulin constantdomain sequence, however N-terminal fusions are also possible.Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion or bindingcharacteristics of the PTP λ-immunoglobulin chimeras.

In a preferred embodiment, the sequence of a native, mature PTP λpolypeptide, or a soluble (transmembrane domain-inactivated) formthereof, is fused to the N-terminus of the C-terminal portion of anantibody (in particular the Fc domain), containing the effectorfunctions of an immunoglobulin, e.g. IgG-1. It is possible to fuse theentire heavy chain constant region to the PTP λ sequence. However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216,taking the first residue of heavy chain constant region to be 114 [Kobetet al., supra], or analogous sites of other immunoglobulins) is used inthe fusion. In a particularly preferred embodiment, the PTP λ sequence(full length or soluble) is fused to the hinge region and CH2 and CH3 orCH1, hinge, CH2 and CH3 domains of an IgG-1, IgG-2, or IgG-3 heavychain. The precise site at which the fusion is made is not critical, andthe optimal site can be determined by routine experimentation.

In some embodiments, the PTP λ-immunoglobulin chimeras are assembled asmultimers, and particularly as homo-dimers or -tetramers (WO 91/08298).Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basic fourunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in multimeric form in serum. In the case ofmultimer, each four unit may be the same or different.

Various exemplary assembled PTP A-immunoglobulin chimeras within thescope herein are schematically diagramed below:

(a) AC_(L) -AC_(L) ;

(b) AC_(H) -[AC_(H), AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H) ];

(c) AC_(L) -AC_(H) -[AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), V_(L) C_(L)-AC_(H), or V_(L) C_(L) -V_(H) C_(H) ];

(d) AC_(L) -V_(H) C_(H) -[AC_(H), or AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H) ];

(e) V_(L) C_(L) -AC_(H) -[AC_(L) -V_(H) C_(H), or V_(L) C_(L) -AC_(H) ];and

(f) [A-Y]_(n) -[V_(L) C_(L) -V_(H) C_(H) ]₂,

wherein

each A represents identical or different novel PTP λ polypeptide aminoacid sequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the PTP λ amino acid sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the PTP λ polypeptide sequences are fused to the 3' end ofan immunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom, H. R. etal., Mol. Immunol. 28, 1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to a PTPλ-immunoglobulin heavy chain fusion polypeptide, or directly fused tothe PTP λ polypeptide. In the former case, DNA encoding animmunoglobulin light chain is typically coexpressed with the DNAencoding the PTP λ-immunoglobulin heavy chain fusion protein. Uponsecretion, the hybrid heavy chain and the light chain will be covalentlyassociated to provide an immunoglobulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodsuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG-1 and IgG-3 immunoglobulinsequences is preferred. A major advantage of using IgG-1 is that IgG-1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG-3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG-3hinge is longer and more flexible, so it can accommodate larger`adhesin` domains that may not fold or function properly when fused toIgG-1. While IgG immunoadhesins are typically mono- or bivalent, otherIg subtypes like IgA and IgM may give rise to dimeric or pentamericstructures, respectively, of the basic Ig homodimer unit. Multimericimmunoadhesins are advantageous in that they can bind their respectivetargets with greater avidity than their IgG-based counterparts. Reportedexamples of such structures are CD4-IgM (Traunecker et al., supra);ICAM-IgM (Martin et al., J. Virol. 67, 3561-68 [1993]); and CD2-IgM(Arulanandam et al., J. Exp. Med. 177, 1439-50 [1993]).

For PTP λ-Ig immunoadhesins, which are designed for in vivo application,the pharmacokinetic properties and the effector functions specified bythe Fc region are important as well. Although IgG-1, IgG-2 and IgG-4 allhave in vivo half-lives of 21 days, their relative potencies atactivating the complement system are different. IgG-4 does not activatecomplement, and IgG-2 is significantly weaker at complement activationthan IgG-1. Moreover, unlike IgG-1, IgG-2 does not bind to Fc receptorson mononuclear cells or neutrophils. While IgG-3 is optimal forcomplement activation, its in vivo half-life is approximately one thirdof the other IgG isotypes. Another important consideration forimmunoadhesins designed to be used as human therapeutics is the numberof allotypic variants of the particular isotype. In general, IgGisotypes with fewer serologically-defined allotypes are preferred. Forexample, IgG-1 has only four serologically-defined allotypic sites, twoof which (G1m and 2) are located in the Fc region; and one of thesesites G1m1, is non-immunogenic. In contrast, there are 12serologically-defined allotypes in IgG-3, all of which are in the Fcregion; only three of these sites (G3m5, 11 and 21) have one allotypewhich is nonimmunogenic. Thus, the potential immunogenicity of a γ3immunoadhesin is greater than that of a γ1 immunoadhesin.

PTP λ-Ig immunoadhesins are most conveniently constructed by fusing thecDNA sequence encoding the PTP λ portion in-frame to an Ig cDNAsequence. However, fusion to genomic Ig fragments can also be used (see,e.g. Gascoigne et al., Proc. Natl. Acad. Sci. USA 84, 2936-2940 [1987];Aruffo et al., Cell 61, 1303-1313 [1990]; Stamenkovic et al., Cell 66,1133-1144 [1991]). The latter type of fusion requires the presence of Igregulatory sequences for expression. cDNAs encoding IgG heavy-chainconstant regions can be isolated based on published sequence from cDNAlibraries derived from spleen or peripheral blood lymphocytes, byhybridization or by polymerase chain reaction (PCR) techniques.

Other derivatives comprise the novel peptides of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyvinylalkylene etherssuch a polyethylene glycol, polypropylene glycol.

The PTP λ polypeptides may be linked to various nonproteinaceouspolymers, such as polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PTP λ polypeptides may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization, incolloidal drug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Oslo, A., Ed. (1980).

H. Anti-PTP λ Antibody Preparation

(i) Polyclonal antibodies

Polyclonal antibodies to a PTP λ molecule generally are raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the PTP λ and an adjuvant. It may be useful to conjugate the PTP λ ora fragment containing the target amino acid sequence to a protein thatis immunogenic in the species to be immunized, e.g. keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg or 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFreud's complete adjuvant by subcutaneous injection at multiple sites. 7to 14 days later the animals are bled and the serum is assayed foranti-PTP λ antibody titer. Animals are boosted until the titer plateaus.Preferably, the animal boosted with the conjugate of the same PTP λ, butconjugated to a different protein and/or through a differentcross-linking reagent. Conjugates also can be made in recombinant cellculture as protein fusions. Also, aggregating agents such as alum areused to enhance the immune response.

(ii) Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-PTP λ monoclonal antibodies of the presentinvention may be made using the hybridoma method first described byKohler & Milstein, Nature 256:495 (1975), or may be made by recombinantDNA methods [Cabilly, et al., U.S. Pat. No. 4,816,567].

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, "chimeric" or "hybrid" antibodies are prepared that have thebinding specificity of an anti-PTP λ monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for a PTP λpolypeptide and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵ I, ³² P, ¹⁴ C, or ³ H, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

(iii) Humanized antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed Aug. 21, 1992,which is a continuation-in-part of application Ser. No. 07/715,272 filedJun. 14, 1991.

Alternatively, it is now possible to produce transgenic animals (e.g.mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA 90, 2551-255(1993); Jakobovits et al., Nature 362, 255-258 (1993).

(iv) Bispecific antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for aPTP λ polypeptide, the other one is for any other antigen. Methods formaking bispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, and second and thirdconstant regions of an immunoglobulin heavy chain (CH2 and CH3). It ispreferred to have the first heavy chain constant region (CH1) containingthe site necessary for light chain binding, present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are cotransfected into a suitable host organism.This provides for great flexibility in adjusting the mutual proportionsof the three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance. In a preferred embodiment of this approach, the bispecificantibodies are composed of a hybrid immunoglobulin heavy chain with afirst binding specificity in one arm, and a hybrid immunoglobulin heavychain-light chain pair (providing a second binding specificity) in theother arm. It was found that this asymmetric structure facilitates theseparation of the desired bispecific compound from unwantedimmunoglobulin chain combinations, as the presence of an immunoglobulinlight chain in only one half of the bispecific molecule provides for afacile way of separation. This approach is disclosed in PCT applicationWO 94/04690 published Mar. 3, 1994.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(v) Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

I. Peptide and Non-Peptide Analogs of PTP λ Polypeptides

Peptide analogs of the PTP λ polypeptides of the present invention aremodeled based upon the three-dimensional structure of the nativepolypeptides. Peptides may be synthesized by well known techniques suchas the solid-phase synthetic techniques initially described inMerrifield, J. Am. Chem. Soc. 15, 2149-2154 (1963). Other peptidesynthesis techniques are, for examples, described in Bodanszky et al.,Peptide Synthesis, John Wiley & Sons, 2nd Ed., 1976, as well as in otherreference books readily available for those skilled in the art. Asummary of peptide synthesis techniques may be found in Stuart andYoung, Solid Phase Peptide Synthelia, Pierce Chemical Company, Rockford,Ill. (1984). Peptides may also be prepared by recombinant DNAtechnology, using a DNA sequence encoding the desired peptide.

In addition to peptide analogs, the present invention also contemplatesnon-peptide (e.g. organic) compounds which display substantially thesame surface as the peptide analogs of the present invention, andtherefore interact with other molecules in a similar fashion.

J. Use of the PTP λ Polypeptides

The PTP λ polypeptides of the present invention are useful for a varietyof purposes. For example, the PTP λ polypeptides of the presentinvention are useful in the identification and purification of the PTP λligand, for which a possible location is the brain. The purification maybe performed by using the native receptor(s) or immunoadhesins,comprising a fusion of the extracellular domain of the receptor(s) to animmunoglobulin heavy chain constant region. The ligands are expected tobe useful in the treatment of paralytic-type diseases.

An increased level of expression of the PTP λ receptors of the presentinvention may be useful in reducing metastasis of various tumors of thelung and other organs. The expression of the receptor may be upregulatedby anti-PTP λ antibodies, which are capable of cross-linking and therebyactivating the receptors. Non-antibody cross-liking agents may also beemployed for this purpose.

The PTP λ polypeptides of the present invention are also useful asmolecular markers of the tissues in which they are specificallyexpressed. As such, the PTP λ polypeptide is useful for tissue typing ofspecific mammalian tissues.

Native PTP λ polypeptides and their functional equivalents are alsouseful in screening assays designed to identify agonists or antagonistsof native PTP λ polypeptides. Such assays may take the form of anyconventional cell-type or biochemical binding assay, and can beperformed in a variety of assay formats well known to those skilled inthe art. An example is the so called "two-hybrid" assay format using theMatchmaker Two-Hybrid System (Clontech) according to the manufacturersinstructions.

The native PTP λ polypeptides of the present invention are also usefulas protein molecular weight markers for protein gels.

Nucleic acids encoding the PTP λ polypeptides of the present inventionare also useful in providing hybridization probes for searching cDNA andgenomic libraries for the coding sequence of other PTP λ polypeptidesanalogs in other species.

Antagonists of the PTP λ polypeptide of the present invention are usefulfor inhibiting the biological activity of the enzyme, thereby inhibitingthe biological effects of tyrosine dephosphorylation. Agonists of thePTP λ polypeptide are useful for increasing or simulating the biologicaleffects of the native PTP λ polypeptide.

K. Materials and Methods

1. RNA Isolation and Polymerase Chain Reaction

Messenger RNA was isolated from the non-adherent Lin^(lo) CD34^(hi)fraction of fetal yolk sac cells (Micro-FastTrack, InVitrogene). Poly A+RNA was reverse transcribed with random hexamers (Promega) and Molonymurine Leukemia virus reverse transcriptase (SuperScript II, GIBCO BRL).One quarter of this cDNA was amplified by PCR using degenerate mixedoligonucleotide primers. Sense and anti-sense primers corresponding tothe amino acid sequences (H/D)FWRM(I/V)W (SEQ ID NO:5)(5'-A(C/T)TT(C/T)TGG(A/C)GIATG(A/G)TITGG-3') (SEQ ID NO:6) andWPD(F/H)GVP (SEQ ID NO:7) (5'-GGIAC(G/A)(T/A)(G/A)(G/A)TCIG GCCA-3')(SEQ ID NO:8) respectively were used. PCR was carried out in 1× Taq DNApolymerase buffer (GIBCO BRL) plus 0.2 mM of each dNTP, 10% DMSO and 5units Taq polymerase (GIBCO BRL) for 25 cycles of 94° C. for 1 minute,55° C. for 1 minute and 72° C. for 1 minute. The PCR products weretreated with Klenow enzyme (New England Biolabs) at 30° C. for 30minutes, cloned into the Smal site of the pRK-5 plasmid (Genentech,Inc.) and subsequently sequenced (Sequenase, USB).

2. Isolation of cDNA clones

Adapter-linked double stranded cDNA was prepared from A+ RNA of day-10mice embryos (Marathon-ready cDNA synthesize kit, Clontech) using eitherrandom hexamer or oligo dT primers. Full-length cDNA was isolated by 5'or 3' rapid amplification of cDNA ends (RACE) of the marathon-readycDNAs. A lambda cDNA library of adult mouse lung was screened followingthe standard protocol using cDNA fragments isolated by RACE as probes.

3. Bacterial Expression of GST-PTP Fusion Protein

cDNA sequences encoding amino acids 791 to 1436 or amino acids 43 to 741containing either the cytoplasmic region or the extracellular region ofPTP λ was obtained by PCR. PCR fragments were then treated with SalI andNotI restriction enzymes and cloned into the pGEX-4T-1 plasmid(Pharmacia). Fusion proteins were affinity purified using Glutathionesepharose columns (Pharmacia). Polyclonal anti-serum against either thecytoplasmic (Cy) or extracellular (Ex) region was generated byimmunizing rabbits with each purified GST-fusion protein.

4. Indirect Immunofluorescence of PC-12 Cells

NGF-treated or untreated PC-12 cells grown on cover slips were fixedwith 4% formaldehyde and 0.1% Triton X-100 in phosphate-buffered saline(PBS) and permeabilized with 0.05% saponin. Fixed cells were thenblocked with 10% normal goat serum plus 0.05% NP40 in PBS, incubatedwith polyclonal rabbit anti-Cy primary antiserum (1:3000 dilution),washed, and incubated with phycoerytherin (PE)-tagged goat antibody torabbit immunoglobulin G. Cells were viewed and digital images were takenby fluorescence confocal microscopy.

5. Immunoprecipitation and Tyrosine Phosphatase Assay of PTPλ

PC-12 cells expressing endogenous PTP λ were washed in cold PBS, thenlysed in buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 1 mM DTT, 1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/mlaprotinin, 10 mM NaF, 0.5 mM okadaic acid, 10% (v/v) glycerol, 1% (v/v)Triton X-100, 0.5% (w/v) sodium deoxycholate and 0.01% (w/v) SDS(AUTHOR?, EMBO J., 13(16):3763-3771 (1994)). Cell lysates wereprecleared by incubating with 50 ml of washed protein A-Sepharose beads(Pharmacia). Precleared lysate was then incubated with proteinA-Sepharose beads pre-coupled with rabbit polyclonal antiserum (20 mlserum/50 ml beads) at 40° C. for 15 hours. The protein A-Sepharose/PTP λimmunoprecipitate complex was then processed as described (Jiang et al.,Mol. Cell Biol. 13(5):2942-2951 (1993)). Briefly, the complex was washedfour times with HNTG buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10%glycerol, 0.1% Triton X-100) and once with M7.6 buffer (60 mM Tris-HCl,pH 7.6, 5 mM EDTA, 10 mM DTT, 50 mM NaCl, 50 mg/ml bovine serumalbumin). Washed immunoprecipitate complex was resuspended in M7.6buffer and subject to non-radioactive protein tyrosine phosphatase assaywith synthetic oligopeptide substrates (PPS1 corresponds to the hirudin53-63 C-terminal fragment: Biotin-DGDFEEIPEEY-PO₄ (SEQ ID NO:9), PPS2corresponds to amino acids 1-17 of human gastrin:Biotin-EGPWLEEEEEAY-PO₄ (SEQ ID NO:10)). PTPase assay was carried outfollowing the manufacturer's procedures (Tyrosine Phosphatase Assay Kit,Boehringer Mannheim).

6. Northern Analysis

A 2.5 kb cDNA fragment encoding the cytoplasmic region of PTP λ was usedto probe the murine multi-tissue northern blots (Clontech) or the A+ RNAof PC-12 cells.

7. In Situ Hybridization

Rat E15.5 embryos and P1 brains were immersion fixed overnight at 4° C.in 4% paraformaldehyde, then cryoprotected overnight in 15% sucrose.Adult rat brains were fresh frozen with powdered dry ice. All tissueswere sectioned at 16 um, and processed for in-situ hybridization for PTPλ using ³³ P-UTP labeled RNA probes. Sense and antisense probes weresynthesized from a 2.5 kb DNA fragment of PTP λ using SP6 or T7polymerase, respectively.

Further experimental details will be apparent from the followingnon-limiting examples.

L. Examples

Example1--Isolation and Characterization of the cDNA encoding PTP λ

In order to isolate novel receptor protein tyrosine phosphatases (PTPs)expressed in murine primitive hematopoietic cells, we undertook thecloning of PCR fragments produced by priming with sequences directedagainst conserved protein motifs found in PTPs from a number ofdifferent genes and species (Dixon, Ann. Ny. Acad. Sci. 766:18-22(1995)). Analysis of 70 different PCR-derived subclones revealed anarray of previously described PTPs, as well as two novel PTPs. One ofthese novel PTPs, termed PTP HSC, is a member of the PTP PEST family ofenzymes, and it has been previously described (Cheng et al., Blood, inpress). The second novel PCR fragment was homologous to PTPs κ and μ,both related receptor-type PTPs that mediate homophilic adhesion(Brady-Kalnay et al., Curr. Opin. Cell. Biol. 7(5):650-657 (1995)).

In order to further characterize the cDNA encoding this novel PTP, acombined cloning approach that utilized RACE as well as cloning fromphage cDNA libraries was performed. The composite cDNA (SEQ ID NO:1) andderived protein (SEQ ID NO:2) sequences determined from these variousclones is shown in FIGS. 1A-1D. The ATG start codon utilized fortranslation of this large open reading frame was embedded within aconsensus Kozak sequence, and there are several translational stopcodons upstream of this initiator codon. As can be seen from FIGS.1A-1D, the protein (SEQ ID NO:2) derived from this cDNA (SEQ ID NO:1) isa large receptor-like molecule of 1,436 amino acids and a molecularweight of approximately 161,176 daltons.

FIGS. 2A-2B illustrate that the novel, hematopoietically-derived PTP-related protein reported here shows a high degree of homology to bothPTP κ (.sup.˜ 60%) and PTP μ (.sup.˜ 53%) throughout their entirelengths (Jiang et al., (1993) supra and Gebbink et al., (1991) supra).Because this novel PTP is homologous throughout its entire length toPTPs κ and μ, it appears that the new PTP polypeptide contains MAM, IgG,4 fibronectin type III, and two cytoplasmically localized phosphatasedomains (see FIGS. 2A-2B) (Brady-Kalnay et al., Curr. Opin. Cell. Biol.7(5):650-657 (1995), Jiang et al., (1993) supra and Gebbink et al.,(1991) supra). These homologies with the novel PTP polypeptide aresomewhat less than the homology between PTPκ and μ (.sup.˜ 62%),suggesting that the novel PTP polypeptide reported here is rather moredistantly related to these two PTPs than they are to each other. Thesedata suggest that this novel PTP is the third member of thehomotypically interacting PTP family containing PTPs κ and μ, and wehave therefore named the novel receptor PTP λ.

As can be seen from FIG. 3, the relative sequence homologies in each ofthe domains of these three enzymes suggests that they are indeed closelyrelated. Interestingly, previous data suggested that both the MAM andIgG domains mediated specific homotypic adhesion between PTPs κ and μ(Brady-Kalnay et al., (1994) supra and Zondag et al., (1995) supra), andit is clear from the sequence comparisons between these three relatedproteins that these two domains are substantially homologous. However,the fact that there are a large number of sequence changes between thesetwo motifs is also consistent with the supposition that they can mediatespecific homotypic interactions. Thus, it is likely that, while thesemotifs are undoubtedly structurally related, differences in theirrelative sequences are involved with homotypic recognition.

The overall sequence homologies between the three proteins is alsorelatively high in the FnIII domains, although the homology in the firstof these domains is significantly higher than in the others. Previouswork has also demonstrated that a juxtamembrane site between thetransmembrane domain and the first phosphatase domain is distantlyhomologous to a similar region in the cadherins (Brady-Kalnay et al., J.Cell. Biol. 130(4):977-986 (1995)), and this site shows a high degree ofhomology between these three receptors. A high degree of sequencehomology is also found between the first PTPase domains of these threereceptors, with a somewhat lower level of homology between the secondPTPase domains of these proteins. This latter result may be significant,since it has been reported that the first phosphatase domain is the mostimportant enzymatic motif of the dual phosphatase regions in thereceptor PTPs (Pot et al., J. Biol. Chem. 266(29):19688-19696 (1991)).The homology between these PTPase domains includes many of the residuespreviously found to be important for substrate recognition and tyrosinedephosphorylation in the PTP 1B (Jia et al., Science 268(5218):1754-1758(1995)), although not all of these residues are completely conserved. Insummary, the sequence homologies between these three proteins suggest acommon ancestor as well as potentially similar functions.

Example 2--Analysis of the Enzymatic Activity of PTP λ

In order to analyze the enzymatic activity of the PTPase domains of thenovel PTP λ polypeptide, we immunoprecipitated the enzyme from PC 12cells which we show below express the protein. In these experiments, apolyclonal antibody directed against the entire cytoplasmic domain aspredicted from the cDNA sequence was produced by injecting rabbits witha GST fusion containing this region of the receptor. Theimmunoprecipitate was incubated with a tyrosine phosphorylated peptideusing a commercial kit, and the degree of dephosphorylation wasdetermined using an anti-phosphotyrosine antibody. As is shown in FIG.4, the immunoprecipitate obtained using the immune serum had clearphosphatase activity, while the preimmune serum immunoprecipitate showedno such activity. In addition, FIG. 4 demonstrates that this enzymaticactivity was completely inhibited by the inclusion of vanadate, a potenttyrosine phosphatase inhibitor. Thus, the PTP λ polypeptide encoded bythe cDNA (SEQ ID NO:1) described herein and shown in FIGS. 1A-1D isclearly a receptor tyrosine phosphatase protein.

Example 3--Tissue Expression of the PTP λ Transcript

As is shown in FIG. 5, northern blot analysis of fetal as well as adulttissues demonstrates that PTP λ mRNA is expressed in a diversity oftissues outside of the hematopoietic progenitor cells from which it wasoriginally cloned. Thus, the expression of PTP λ mRNA is detectedthroughout embryonic development beginning in the very early embryo atday 7. Interestingly, analysis of adult organs reveals that the PTP λtranscript is expressed specifically in only a subset of tissues. Thus,there appears to be a very high level of expression of the PTP λpolypeptide in adult brain, lung and kidney, a much decreased level inheart, skeletal muscle and testis, and a lack of obvious expression atthis exposure in spleen and liver.

The high level of PTP λ expression in lung and brain, together with thelack of expression in liver, is in contrast to PTP κ, a PTP which isexpressed at high levels in liver but is almost undetectable in lung andbrain (Jiang et al., (1993) supra). Thus, in spite of the fact that PTPκ was originally isolated from hematopoietic stem cells, there is noobvious expression in two sites which contain hematopoietic cells, thespleen and the liver. The lack of signal in the spleen, an organ whichcontains mostly mature hematopoietic cells, suggests, therefore, thatthis receptor may be expressed specifically in earlier hematopoieticprogenitor cells. interestingly, there appears to also be analternatively spliced transcript in the lung which is not detected inthe other two organs that express this receptor at high levels nor inthe embryos although the nature of this alternatively spliced transcriptremains to be determined. In summary, these data demonstrate that PTP λis specifically expressed in a subset of adult tissues, some of whichare divergent from PTP κ.

Example 4--In Situ Hybridization Analysis

We performed in-situ mRNA analysis of the rat E15.5 embryo, P1 and adultrat brain to determine potential sites of PTP λ production. The resultsin FIG. 6 shown that extensive PTP λ expression was observed indeveloping skeletal, epithelial, and neuronal structures throughout theE15.5 embryo. Systemic expression was observed in various developingskeletal elements such as vertebral perichondrium, intervertebral discs,teeth, mandible and maxilla (FIG. 6, Panels A and B). Expression of PTPλ within urogenital structures included the genital tubercle (FIG. 6,Panels A and B), urethra, and urogenital sinus (not shown). Otherpositive areas of PTP λ expression included the anal canal (not shown),skin, olfactory and oral epithelium, esophagus (FIG. 6, Panels A and B),pituitary (FIG. 6, Panels A, B and C), aura mater (FIG. 6, Panels A, Band D), kidney (FIG. 6, Panels A and B), and lung (FIG. 6, Panels A andB). Higher magnification reveals expression restricted to developingglomeruli in the cortical region of the kidney (FIG. 6, Panels F and G),and bronchiolar epithelium of the lung (FIG. 6, Panels H and I). Withinthe E15.5 embryonic nervous system, high levels of expression wereobserved in the developing cerebral cortex (FIG. 6, Panels A and B),floor of the midbrain, choroid plexus primordium, gigantocellularreticular nucleus of the brain stem (FIG. 6, Panels A, B and C), auramater and spinal cord (FIG. 6, Panels A, B and D). High magnification ofthe spinal cord reveals highest expression of PTP λ in the ventrolateralmotor column (FIG. 6, Panel D).

In P1 and adult brain, expression of PTP λ was localized to regionsderived from embryonic anlage that also contained high levels ofexpression. For instance, expression in the embryonic midbrain precededthe high levels of PTP λ expression in the P1 and adult substantia nigra(FIG. 7, Panels C and E, respectively). Expression in the embryonicforebrain (FIG. 6, Panel A) preceded expression observed in the innerlayers of the P1 and adult cortex (FIG. 7, Panels A, B and D, E,respectively). Expression in the choroid plexus primordia of the embryobegets high levels of expression in the P1 brain (FIG. 7, Panel A), andlow levels of expression in the adult brain (FIG. 7, Panel D).

In general, PTP λ expression in the adult brain appears to bedownregulated relative to the P1 brain (FIG. 7). However, other areas ofprominent expression in both P1 and adult brain include piriform cortexand endopiriform nucleus (FIG. 7, Panels A and D, respectively),amygdaloid nuclei, subiculum, and CAT, CA2 and, to a lesser extent, CA3of the hippocampal formation (FIG. 7, Panels B and E, respectively). TheP1 brain also exhibits strong expression throughout the septal area,basal ganglia, thalamus, and midbrain (FIG. 7, Panels A, B and C). Weakexpression is observed in the adult superior colliculus as well asscattered expression throughout the thalamus (FIG. 7, Panel E).

Example 5--Expression of PTP λ in PC 12 Cells

The expression of PTP λ in various regions throughout the embryonic,neonatal and adult brain suggested that this receptor might be expressedin PC12 cells, a cell line which is derived from a neuralpheochromocytoma. Indeed, the immunoprecipitation experiments describedin Example 2 above demonstrated enzymatic activity in anti-PTP λprecipitates derived from these cells. In addition, these cells willdifferentiate and extend neurites in response to nerve growth factor, sothey provided a system to test a possible role for PTP λ in thisdevelopmental transition. As is shown in FIG. 8, the novel PTP λreceptor polypeptide is indeed expressed in these neuronal progenitorcells. FIG. 8 also illustrates that treatment of these cells with NGFresults in a modest upregulation (.sup.˜ 5 fold) of the transcriptencoding this receptor with relatively slow kinetics. These data arethus consistent with a role for this receptor in some aspect of neuronaldifferentiation in this cell line.

In order to investigate the distribution of PTP λ on PC12 cells,immunofluorescence was performed using cells that were left untreated orwere treated with NGF to induce neurite outgrowth stained with anantibody directed against the cytoplasmic domain of the PTP λ receptor.As is shown in FIG. 9, PTP λ is expressed at significant levels in bothtreated and untreated cells, confirming the enzymatic analysis shown inFIG. 4 and the northern blot analysis shown in FIG. 8. Perhaps moreinteresting, however, is the cellular distribution of the PTP λpolypeptide. As FIG. 9 shows, PTP λ is found to be partitioned on theneurites as well as on the growth cone-like structures at the neuritetips. These data are consistent with a role for this receptor in neuritefunction, perhaps analogous to that recently described for two differentDrosophila receptor PTPs (Desai et al., supra and Kreuger et al.,supra).

M. Discussion

The relative levels of tyrosine phosphorylation of a diversity ofproteins is critical for the regulation of a number of activities duringembryonic differentiation and throughout the life of the mammalianorganism. The absolute levels of this modification are mediated throughthe balance of the enzymatic activities of tyrosine kinases with thoseof the tyrosine phosphatases. In both cases, these large families ofproteins perform their roles through conserved enzymatic domains thatare coupled to a plethora of specificity-determining motifs. Thesevarious motifs are found in the context of both membrane traversing,receptor-like molecules as well as intracellular forms of the enzymes.

The similarities in overall structure of the tyrosine kinases andtyrosine phosphatases suggest that they mediate their relative specificactivities through the use of these various domains. In the cases ofsome of the receptor PTPs, the extracellular motifs are somewhat unusualin that they contain highly glycosylated regions with currently unknownligand specificity. Alternatively, a subset of thesereceptor-phosphatases also contain a diversity of domains, includingimmunoglobulin-like and fibronectin-like, which are associated with celladhesion and ligand binding activities in other protein families. Amongthe most interesting of these types of adhesion-associated PTPs are theκ and μ receptors which are involved with homotypic types ofinteractions. Earlier predictions, based upon the likely function ofthese receptors in mediating cell adhesion as well as their limitedtissue distribution, suggested that there might be other κ and μ-likereceptor PTPs with different tissue dispositions. We here report theisolation of the third member of this family of homotypicallyinteracting receptor PTPs, PTP λ, which may be associated with theconstruction of epithelial and neural structures during development andin the adult.

The strongest data suggesting that the novel PTP λ polypeptide describedherein is homologous to the κ and μ receptors lies in the high degree ofsequence conservation between these three proteins. Analysis of thesethree receptors clearly revealed that the novel PTP λ polypeptide of thepresent invention had a high degree of sequence homology with PTP κ andPTP μ throughout the entire length of the proteins. This homologyincluded the four major types of domains contained in this familyincluding the MAM, the immunoglobulin (IgG), the fibronectin type III(FN III) and the dual phosphatase (PTPase) domains (Jiang et al., (1993)supra and Gebbink et al., (1991) supra). Because previous data havesuggested that both the MAM as well as the IgG domain appear to beinvolved with homotypic adhesion (Brady-Kalnay et a., (1994) supra andZondag et al., supra), it is likely that these motifs are used for asimilar function in PTP λ, a hypothesis that is consistent with a rolefor this receptor in cell adhesion. However, the degree of sequencehomology of these domains between the herein reported PTP λ receptor andthe PTP κ and PTP μ receptors is quite divergent, suggesting that thenovel receptor may also specifically mediate a homophilic interactiononly to itself and not to these domains in the other family members(Zondag et al., supra). As will be discussed below, these results,together with the tissue localization of this receptor, suggest that itmay be involved with the formation of very specific edifices duringdevelopment. It will, of course, be interesting to determine thestructural aspects of these domains which are involved with homophilicbinding, especially in light of the recent crystallographic analysis ofone of the homophilically interacting cadherins (Shapiro et al., Nature374(6520):327-337 (1995)). While it is difficult to currently interpretthe significance of the conservation of the FNIII domains, which may actas spacer domains to extend the functionally critical MAM and IgGdomains from the cell surface, the conservation of the dual PTP domainslends itself to some comment. For example, the higher degree ofconservation of the first domain as compared to the second substantiatesprevious work suggesting that the N-terminal PTPase motif is theenzymatically active one, while the C-terminal domain may be involvedwith the regulation of enzyme activity (Pot et al., supra). We haveattempted, without success, to bacterially express enzymatically activeforms of the PTPase domains of PTP λ under conditions which gave a highlevel of activity with another PTP, the PTP HSC (Cheng et al., supra)(J. Cheng and L. Lasky-unpublished observations). These negative data,which of course might be technical, suggest that the PTP λ polypeptidemay require an activation event, although it is clear from theimmunoprecipitation studies with PC 12 cells that this receptor isendowed with enzymatic activity. Finally, previous data have suggested arole for this category of receptor PTPs in cadherin/catenin regulation,and other investigators have pointed to an intracellular, juxtamembranesite with significant homology to a similarly localized region in thecadherins (Brady-Kalnay et al., Curr. Opin. Cell. Biol. 7(5):650-657(1995) and Brady-Kalnay et al., J. Cell. Biol. 130(4):977-986 (1995)).We have also found a very high degree of sequence conservation in thisregion, again consistent with a potential role for this domain incadherin interactions. In summary, the data reported here are consistentwith PTP λ being the third member of the homotypically interactingreceptor PTP family.

The in situ hybridization analysis of the expression of PTP λ in thedeveloping embryo and adult suggest some potentially importanthypotheses. The expression of this receptor in a diversity of developingskeletal areas as well as in epithelial sites which line various organsystems with a layer of these cells, coupled with the proposed role forPTP μ (Brady-Kalnay et al., Curr. Opin. Cell. Biol. 7(5):650-657 (1995)and Brady-Kalnay et al., J. Cell. Biol. 130(4):977-986 (1995)), andpotentially PTP κ, in the control of cadherin adhesion suggests that thenovel PTP λ might be involved in a similar type of adhesion control inthe developing embryo. For example, the development of epithelial layersin the lung bronchioles and kidney glomeruli requires that a sheet ofepithelial cells that is one cell thick be constructed. Thus, as thecells grow and migrate during embryogenesis, they would require amechanism where they sensed the location of other epithelial cells thatwere in contact with them, so that this cellular contiguity initiated anadhesive response that inhibited further epithelial movement via theenhancement of cell adhesion. One mechanism that would provide for sucha sensing phenomenon would be that proposed by Tonks and colleagues(Brady-Kalnay et al., Curr. Opin. Cell. Biol. 7(5):650-657 (1995) andBrady-Kalnay et al., J. Cell. Biol. 130(4):977-986 (1995)). In thishypothesis, the μ receptor PTP comes into homophilic contact withanother μ receptor PTP on an adjacent cell, and this contact upregulatescadherin-mediated adhesion through the dephosphorylation of thecadherin/catenin complex. The formation of single cell-thick epithelialstructures in these embryonic organs could be mediated by a similar typeof sensing mechanism using PTP κ. The expression of this receptor PTP inbone forming chondrocytes would also be expected to perform a similartype of sensing and adhesion function to assemble these structures,although this type of anatomy, which is more complex than thethin-walled epithelial-like morphology described above, would beexpected to involve more elaborate types of sensing and adhesivemechanisms. Finally, because many common types of tumors of the lung andother organs involve epithelial cells, it is possible that disruptionsin the proposed function of this type of adhesion sensing mechanismmight be involved with the disorganized morphology and high rate ofmetastasis of these tumors (Kemler, supra and Beherens et al., supra).Together, these hypotheses suggest a critical role for PTP λ in theformation of various epithelial-like structures in the embryo.

Recent data from the Drosophila system also suggest interestingpossibilities for the function of PTP λ in the developing nervous system(Desai et al., supra and Kreuger et al., supra). In these reports, threedifferent Drosophila receptor PTPs, termed DPTP69D, DPTP99A and DLAR,which all contain IgG and fibronectin type III adhesion domains similarto those found in PTP λ, were shown to be critically involved withneuronal pathfinding in the developing nervous systems. Thus, mutationsin either of these receptors resulted in a loss of the ability ofcertain neural subsets to become reoriented during their formation inthe embryo. Because PTP λ is expressed in a number of developing neuralsites, it is possible that it plays a similar role in the pathfinding ofnerves in mammals. Thus, the expression of this PTP in the developingmidbrain, forebrain, and other neural sites would dispose it to functionas a mediator of pathfinding in these maturing systems. Interestingly,the expression of this receptor in these embryonic anlage was confirmedby expression in the adult sites which arise from these embryonicstructures. However, the expression in the adult appeared to be somewhatreduced as compared to that observed in the embryo, and it was far moreorganized. These data suggest that this enzyme might be utilized duringadult neuronal formation, although the apparent decrease in adultexpression suggests a potentially more critical role duringembryogenesis. The observed expression of this receptor in neuronalprogenitor PC 12 cells, coupled with the upregulation of the transcriptduring neurite formation in response to NGF in these cells, also agreeswith a role for this receptor PTP during neural pathfinding. Indeed, theobservation that this PTP is expressed on neurites as well as on thegrowth-cone like structures at the tips of these processes is consistentwith a potential role for this receptor in neuronal pathfinding in themammalian nervous system. However, the relatively slow kinetics ofupregulation suggest that this may be a late function. Finally, whilethe clear observation of the loss of pathfinding in Drosophila will bedifficult to recapitulate in the mouse, due to the relatively highcomplexity of the mammalian nervous system, it will nevertheless bepotentially of interest to examine the formation of the nervous systemin animals which have been made null for the expression of thisreceptor.

In summary, the data reported herein demonstrate the existence of athird member of the family of receptor PTPs, PTP λ, that appear to beinvolved with homotypic adhesion and, potentially, cadherin mediatedorgan formation. The role that this novel receptor might play in theformation of epithelial sheets and neuronal structures remains to bedetermined. However, the existence of three of these types of receptorsfurther suggests that this growing family may be involved with thespecific formation of various types of complex structures duringdevelopment as well as in the adult.

N. Concluding Remarks

The foregoing description details specific methods which can be employedto practice the present invention. Having detailed such specificmethods, those skilled in the art will well enough known how to devisealternative reliable methods at arriving at the same information inusing the fruits of the present invention. Thus, however, detailed theforegoing may appear in text, it should not be construed as limiting theoverall scope thereof; rather, the ambit of the present invention is tobe determined only by the lawful construction of the appended claims.All documents cited herein are expressly incorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5769 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 379..4686                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GTTGACTACT CAGCTGCCAG AACATCCAAT CTGGCTCCTG CAACTTTAGA CC -            #AACATATT     60                                                                 - - GTGTTTGATC TTCTCCTGAA CAACTTGGGA GATACGTCTG ATCTTCAGCT TG -            #GTACATAC    120                                                                 - - AGTTGCGCAG TGAATGGCAC TTACGTGTTC ATTGTGCACA TGCTAAAGCT GG -            #CATGATTA    180                                                                 - - ATGTTCGACT GCTATGTCAA CCTGATTAAC AATGAGGATG TCTTGGTGTC AG -            #CTATGCCA    240                                                                 - - ACGATGGTGC TCCAGACCGG CGCCAGTCCC GCTCCGCGCG GCACTGTCCA CT -            #ACGGCTCC    300                                                                 - - CGCTCGCCTT GGGCTCCCGG TCGGGCTCCG GAGGCGTCGC CTCCCCAGCT GC -            #GGGTCTCC    360                                                                 - - AGGACCTAGG CGGCGGCC ATG GCC CGG GCT CAG GCT CTG - #GTC CTG GCG CTC          411                                                                                         - #  Met Ala Arg Ala Gln Ala Leu Val Leu - #Ala Leu                           - #    1              - # 5                 - # 10           - - ACC TTC CAG TTC TGC GCG CCT GAG ACC GAG AC - #T CCC GCA GCT GGC TGC          459                                                                       Thr Phe Gln Phe Cys Ala Pro Glu Thr Glu Th - #r Pro Ala Ala Gly Cys                        15     - #             20     - #             25                  - - ACC TTC GAG GAG GCG AGT GAC CCG GTC GTG CC - #C TGC GAG TTC AGC CAG          507                                                                       Thr Phe Glu Glu Ala Ser Asp Pro Val Val Pr - #o Cys Glu Phe Ser Gln                    30         - #         35         - #         40                      - - GCT CAG TAT GAC GAC TTC CAA TGG GAG CAA GT - #G CGG ATC CAC CCC GGC          555                                                                       Ala Gln Tyr Asp Asp Phe Gln Trp Glu Gln Va - #l Arg Ile His Pro Gly                45             - #     50             - #     55                          - - ACC CGG ACC CCT GAA GAC CTG CCC CAT GGT GC - #C TAC TTG ATG GTC AAT          603                                                                       Thr Arg Thr Pro Glu Asp Leu Pro His Gly Al - #a Tyr Leu Met Val Asn            60                 - # 65                 - # 70                 - # 75       - - GCT TCT CAG CAT ACC CCA GGT CAG AGG GCC CA - #C ATC ATC TTC CAG ACC          651                                                                       Ala Ser Gln His Thr Pro Gly Gln Arg Ala Hi - #s Ile Ile Phe Gln Thr                            80 - #                 85 - #                 90              - - CTG AGC GAG AAC GAC ACC CAT TGT GTG CAG TT - #C AGC TAC TTC CTG TAC          699                                                                       Leu Ser Glu Asn Asp Thr His Cys Val Gln Ph - #e Ser Tyr Phe Leu Tyr                        95     - #            100     - #            105                  - - AGC AGG GAT GGG CAC AGC CCA GGC ACC CTG GG - #G GTC TAC GTG CGC GTG          747                                                                       Ser Arg Asp Gly His Ser Pro Gly Thr Leu Gl - #y Val Tyr Val Arg Val                   110          - #       115          - #       120                      - - AAT GGG GGC CCT CTG GGC AGT GCC GTG TGG AA - #T ATG ACC GGA TCC CAC          795                                                                       Asn Gly Gly Pro Leu Gly Ser Ala Val Trp As - #n Met Thr Gly Ser His               125              - #   130              - #   135                          - - GGC CGT CAG TGG CAC CAG GCT GAG CTG GCT GT - #C AGC ACC TTC TGG CCC          843                                                                       Gly Arg Gln Trp His Gln Ala Glu Leu Ala Va - #l Ser Thr Phe Trp Pro           140                 1 - #45                 1 - #50                 1 -      #55                                                                              - - AAT GAG TWT CAG GTG CTG TTT GAG GCC CTC AT - #C TCC CCA GAC CAC        AAG      891                                                                    Asn Glu Xaa Gln Val Leu Phe Glu Ala Leu Il - #e Ser Pro Asp His Lys                          160  - #               165  - #               170              - - GGC TAC ATA GGC TTA GAC GAC ATC TTG CTC TT - #C AGC TAT CCC TGC GCA          939                                                                       Gly Tyr Ile Gly Leu Asp Asp Ile Leu Leu Ph - #e Ser Tyr Pro Cys Ala                       175      - #           180      - #           185                  - - AAG GCC CCT CAC TTC TCC CGC CTT GGG GAC GT - #G GAG GTC AAT GCA GGC          987                                                                       Lys Ala Pro His Phe Ser Arg Leu Gly Asp Va - #l Glu Val Asn Ala Gly                   190          - #       195          - #       200                      - - CAG AAC GCA TCC TTC CAA TGC ATG GCA GCA GG - #C AGA GCC GCA GAG GCA         1035                                                                       Gln Asn Ala Ser Phe Gln Cys Met Ala Ala Gl - #y Arg Ala Ala Glu Ala               205              - #   210              - #   215                          - - GAA CAC TTC TTC CTG CAG CGT CAG AGT GGA GT - #G CTG GTG CCT GCG GCC         1083                                                                       Glu His Phe Phe Leu Gln Arg Gln Ser Gly Va - #l Leu Val Pro Ala Ala           220                 2 - #25                 2 - #30                 2 -      #35                                                                              - - GGG GTG CGG CAC ATC AGT CAC CGT CGC TTC CT - #G GCC ACT TTT CCG        CTG     1131                                                                    Gly Val Arg His Ile Ser His Arg Arg Phe Le - #u Ala Thr Phe Pro Leu                          240  - #               245  - #               250              - - GCC TCG GTA GGC CGC TCA GAG CAG GAT CTG TA - #C CGT TGC GTG TCC CAG         1179                                                                       Ala Ser Val Gly Arg Ser Glu Gln Asp Leu Ty - #r Arg Cys Val Ser Gln                       255      - #           260      - #           265                  - - GCC CCG CGT GGT GCT GGC GTC TCC AAC TTT GC - #A GAG CTC ATC GTC AAA         1227                                                                       Ala Pro Arg Gly Ala Gly Val Ser Asn Phe Al - #a Glu Leu Ile Val Lys                   270          - #       275          - #       280                      - - GAG CCT CCC ACC CCC ATC GCG CCC CCA CAG CT - #G CTG CGT GCA GGC CCC         1275                                                                       Glu Pro Pro Thr Pro Ile Ala Pro Pro Gln Le - #u Leu Arg Ala Gly Pro               285              - #   290              - #   295                          - - ACC TAC CTC ATT ATC CAG CTC AAC ACC AAC TC - #C ATC ATT GGC GAC GGG         1323                                                                       Thr Tyr Leu Ile Ile Gln Leu Asn Thr Asn Se - #r Ile Ile Gly Asp Gly           300                 3 - #05                 3 - #10                 3 -      #15                                                                              - - CCG ATC GTG CGC AAG GAG ATC GAG TAC CGC AT - #G GCA CGG GGC CCG        TGG     1371                                                                    Pro Ile Val Arg Lys Glu Ile Glu Tyr Arg Me - #t Ala Arg Gly Pro Trp                          320  - #               325  - #               330              - - GCC GAG GTG CAC GCT GTC AAC CTG CAR ACC TA - #C AAG CTG TGG CAT CTG         1419                                                                       Ala Glu Val His Ala Val Asn Leu Xaa Thr Ty - #r Lys Leu Trp His Leu                       335      - #           340      - #           345                  - - GAC CCA GAC ACT GAG TAT GAA ATC AGC GTG CT - #G CTC ACA CGC CCG GGA         1467                                                                       Asp Pro Asp Thr Glu Tyr Glu Ile Ser Val Le - #u Leu Thr Arg Pro Gly                   350          - #       355          - #       360                      - - GAT GGA GGC ACA GGC CGC CCT GGG CCA CCA CT - #G ATC AGC CGG ACC AAG         1515                                                                       Asp Gly Gly Thr Gly Arg Pro Gly Pro Pro Le - #u Ile Ser Arg Thr Lys               365              - #   370              - #   375                          - - TGC GCA GAG CCC ACG AGG GCC CCC AAA GGT CT - #G GCT TTT GCT GAG ATC         1563                                                                       Cys Ala Glu Pro Thr Arg Ala Pro Lys Gly Le - #u Ala Phe Ala Glu Ile           380                 3 - #85                 3 - #90                 3 -      #95                                                                              - - CAG GCT CGC CAG CTG ACC CTG CAG TGG GAG CC - #C CTG GGC TAT AAT        GTC     1611                                                                    Gln Ala Arg Gln Leu Thr Leu Gln Trp Glu Pr - #o Leu Gly Tyr Asn Val                          400  - #               405  - #               410              - - ACA CGT TGT CAT ACC TAC GCT GTG TCC CTT TG - #C TAT CGC TAC ACC CTG         1659                                                                       Thr Arg Cys His Thr Tyr Ala Val Ser Leu Cy - #s Tyr Arg Tyr Thr Leu                       415      - #           420      - #           425                  - - GGC GGC AGC CAC AAC CAG ACC ATC CGG GAG TG - #T GTG AAG ATG GAG CGG         1707                                                                       Gly Gly Ser His Asn Gln Thr Ile Arg Glu Cy - #s Val Lys Met Glu Arg                   430          - #       435          - #       440                      - - GGT GCC AGC CGC TAC ACC ATC AAG AAT CTG CT - #G CCA TTC AGA AAC ATC         1755                                                                       Gly Ala Ser Arg Tyr Thr Ile Lys Asn Leu Le - #u Pro Phe Arg Asn Ile               445              - #   450              - #   455                          - - CAC GTG CGT CTG ATT CTC ACA AAC CCT GAG GG - #G CGC AAG GAG GGC AAG         1803                                                                       His Val Arg Leu Ile Leu Thr Asn Pro Glu Gl - #y Arg Lys Glu Gly Lys           460                 4 - #65                 4 - #70                 4 -      #75                                                                              - - GAG GTC ACC TTC CAG ACA GAT GAA GAT GTG CC - #T GGT GGG ATT GCA        GCT     1851                                                                    Glu Val Thr Phe Gln Thr Asp Glu Asp Val Pr - #o Gly Gly Ile Ala Ala                          480  - #               485  - #               490              - - GAG TCC CTA ACC TTC ACT CCA CTG GAG GAC AT - #G ATC TTT CTC AAG TGG         1899                                                                       Glu Ser Leu Thr Phe Thr Pro Leu Glu Asp Me - #t Ile Phe Leu Lys Trp                       495      - #           500      - #           505                  - - GAG GAG CCC CAG GAG CCC AAT GGC CTC ATC AC - #T CAG TAT GAG ATC AGC         1947                                                                       Glu Glu Pro Gln Glu Pro Asn Gly Leu Ile Th - #r Gln Tyr Glu Ile Ser                   510          - #       515          - #       520                      - - TAC CAA AGC ATT GAG TCC TCA GAC CCA GCA GT - #G AAC GTG CCC GGC CCG         1995                                                                       Tyr Gln Ser Ile Glu Ser Ser Asp Pro Ala Va - #l Asn Val Pro Gly Pro               525              - #   530              - #   535                          - - AGA CGC ACC ATC TCC AAA CTC CGG AAT GAG AC - #T TAC CAC GTC TTC TCC         2043                                                                       Arg Arg Thr Ile Ser Lys Leu Arg Asn Glu Th - #r Tyr His Val Phe Ser           540                 5 - #45                 5 - #50                 5 -      #55                                                                              - - AAC CTG CAT CCC GGC ACC ACG TAT CTG TTC TC - #C GTG CGT GCT CGG        ACG     2091                                                                    Asn Leu His Pro Gly Thr Thr Tyr Leu Phe Se - #r Val Arg Ala Arg Thr                          560  - #               565  - #               570              - - AGC AAG GGC TTC GGC CAG GCG GCT CTC ACT GA - #G ATA ACC ACC AAC ATC         2139                                                                       Ser Lys Gly Phe Gly Gln Ala Ala Leu Thr Gl - #u Ile Thr Thr Asn Ile                       575      - #           580      - #           585                  - - TCA GCT CCC AGC TTT GAT TAT GCC GAC ATG CC - #G TCA CCC CTG GGC GAG         2187                                                                       Ser Ala Pro Ser Phe Asp Tyr Ala Asp Met Pr - #o Ser Pro Leu Gly Glu                   590          - #       595          - #       600                      - - TCC GAG AAC ACC ATC ACT GTG CTG TTG AGG CC - #G GCC CAG GGC CGA GGA         2235                                                                       Ser Glu Asn Thr Ile Thr Val Leu Leu Arg Pr - #o Ala Gln Gly Arg Gly               605              - #   610              - #   615                          - - GCC CCC ATC AGC GTC TAC CAG GTG GTT GTG GA - #G GAA GAG CGG CCA CGG         2283                                                                       Ala Pro Ile Ser Val Tyr Gln Val Val Val Gl - #u Glu Glu Arg Pro Arg           620                 6 - #25                 6 - #30                 6 -      #35                                                                              - - CGC TTG CGG CGG GAG CCC GGA GCT CAG GAC TG - #C TTC TCG GTA CCT        CTG     2331                                                                    Arg Leu Arg Arg Glu Pro Gly Ala Gln Asp Cy - #s Phe Ser Val Pro Leu                          640  - #               645  - #               650              - - ACC TTT GAG ACG GCC CTG GCT CGC GGC CTG GT - #G CAC TAC TTT GGG GCT         2379                                                                       Thr Phe Glu Thr Ala Leu Ala Arg Gly Leu Va - #l His Tyr Phe Gly Ala                       655      - #           660      - #           665                  - - GAA CTG GCT GCC AGC AGC CTG CTT GAG GCC AT - #G CCC TTC ACC GTG GGT         2427                                                                       Glu Leu Ala Ala Ser Ser Leu Leu Glu Ala Me - #t Pro Phe Thr Val Gly                   670          - #       675          - #       680                      - - GAC AAC CAG ACC TAT CGT GGC TTC TGG AAC CC - #A CCG CTT GAG CCC AGA         2475                                                                       Asp Asn Gln Thr Tyr Arg Gly Phe Trp Asn Pr - #o Pro Leu Glu Pro Arg               685              - #   690              - #   695                          - - AAG GCC TAT CTC ATC TAT TTC CAG GCA GCA AG - #C CAC CTG AAA GGG GAA         2523                                                                       Lys Ala Tyr Leu Ile Tyr Phe Gln Ala Ala Se - #r His Leu Lys Gly Glu           700                 7 - #05                 7 - #10                 7 -      #15                                                                              - - ACC CGA CTG AAC TGC ATC CGA ATT GCC AGG AA - #A GCT GCG TGC AAG        GAG     2571                                                                    Thr Arg Leu Asn Cys Ile Arg Ile Ala Arg Ly - #s Ala Ala Cys Lys Glu                          720  - #               725  - #               730              - - AGC AAG CGA CCC CTC GAA GTG TCC CAG AGA TC - #G GAG GAG ATG GGG CTC         2619                                                                       Ser Lys Arg Pro Leu Glu Val Ser Gln Arg Se - #r Glu Glu Met Gly Leu                       735      - #           740      - #           745                  - - ATC CTG GGC ATC TGT GCA GGT GGT CTT GCC GT - #C CTC ATT CTC CTC CTG         2667                                                                       Ile Leu Gly Ile Cys Ala Gly Gly Leu Ala Va - #l Leu Ile Leu Leu Leu                   750          - #       755          - #       760                      - - GGG GCC ATC ATT GTC ATC ATC CGC AAA GGG AA - #G CCA GTG AAC ATG ACG         2715                                                                       Gly Ala Ile Ile Val Ile Ile Arg Lys Gly Ly - #s Pro Val Asn Met Thr               765              - #   770              - #   775                          - - AAA GCC ACG GTC AAC TAC CGC CAG GAG AAG AC - #T CAC ATG ATG AGT GCC         2763                                                                       Lys Ala Thr Val Asn Tyr Arg Gln Glu Lys Th - #r His Met Met Ser Ala           780                 7 - #85                 7 - #90                 7 -      #95                                                                              - - GTG GAC CGC AGC TTC ACA GAT CAG AGT ACT CT - #G CAG GAG GAT GAG        CGG     2811                                                                    Val Asp Arg Ser Phe Thr Asp Gln Ser Thr Le - #u Gln Glu Asp Glu Arg                          800  - #               805  - #               810              - - TTG GGT CTG TCC TTT ATG GAT GCT CCT GGC TA - #T AGT CCT CGT GGA GAC         2859                                                                       Leu Gly Leu Ser Phe Met Asp Ala Pro Gly Ty - #r Ser Pro Arg Gly Asp                       815      - #           820      - #           825                  - - CAG CGA AGC GGT GGT GTC ACC GAG GCC AGC AG - #C CTC CTG GGG GGT TCT         2907                                                                       Gln Arg Ser Gly Gly Val Thr Glu Ala Ser Se - #r Leu Leu Gly Gly Ser                   830          - #       835          - #       840                      - - CCA AGG CGC CCA TGC GGC CGG AAG GGT TCT CC - #G TAT CAT ACC GGG CAG         2955                                                                       Pro Arg Arg Pro Cys Gly Arg Lys Gly Ser Pr - #o Tyr His Thr Gly Gln               845              - #   850              - #   855                          - - CTC CAC CCT GCA GTC CGA GTG GCT GAC CTT CT - #A CAG CAC ATC AAC CAG         3003                                                                       Leu His Pro Ala Val Arg Val Ala Asp Leu Le - #u Gln His Ile Asn Gln           860                 8 - #65                 8 - #70                 8 -      #75                                                                              - - ATG AAG ACA GCC GAG GGC TAC GGC TTC AAG CA - #G GAG TAC GAG AGT        TTC     3051                                                                    Met Lys Thr Ala Glu Gly Tyr Gly Phe Lys Gl - #n Glu Tyr Glu Ser Phe                          880  - #               885  - #               890              - - TTT GAG GGC TGG GAC GCC ACC AAG AAG AAA GA - #C AAG CTC AAG GGC GGC         3099                                                                       Phe Glu Gly Trp Asp Ala Thr Lys Lys Lys As - #p Lys Leu Lys Gly Gly                       895      - #           900      - #           905                  - - CGA CAG GAG CCA GTG TCT GCC TAT GAT CGA CA - #C CAT GTG AAA CTA CAC         3147                                                                       Arg Gln Glu Pro Val Ser Ala Tyr Asp Arg Hi - #s His Val Lys Leu His                   910          - #       915          - #       920                      - - CCG ATG CTG GCA GAC CCT GAT GCC GAC TAC AT - #C TCT GCC AAC TAC ATA         3195                                                                       Pro Met Leu Ala Asp Pro Asp Ala Asp Tyr Il - #e Ser Ala Asn Tyr Ile               925              - #   930              - #   935                          - - GAC GGC TAC CAC AGG TCA AAC CAC TTC ATA GC - #C ACT CAA GGG CCA AAG         3243                                                                       Asp Gly Tyr His Arg Ser Asn His Phe Ile Al - #a Thr Gln Gly Pro Lys           940                 9 - #45                 9 - #50                 9 -      #55                                                                              - - CCT GAG ATG ATC TAC GAT TTC TGG CGC ATG GT - #G TGG CAG GAA CAG        TGT     3291                                                                    Pro Glu Met Ile Tyr Asp Phe Trp Arg Met Va - #l Trp Gln Glu Gln Cys                          960  - #               965  - #               970              - - GCG AGC ATC GTC ATG ATC ACC AAG CTG GTA GA - #G GTG GGC AGG GTG AAG         3339                                                                       Ala Ser Ile Val Met Ile Thr Lys Leu Val Gl - #u Val Gly Arg Val Lys                       975      - #           980      - #           985                  - - TGT TCT CGC TAC TGG CCT GAG GAC TCA GAC AT - #G TAT GGG GAC ATC AAG         3387                                                                       Cys Ser Arg Tyr Trp Pro Glu Asp Ser Asp Me - #t Tyr Gly Asp Ile Lys                   990          - #       995          - #       1000                     - - ATC ACG CTG GTA AAG ACA GAG ACA CTG GCT GA - #G TAT GTG GTG CGC ACC         3435                                                                       Ile Thr Leu Val Lys Thr Glu Thr Leu Ala Gl - #u Tyr Val Val Arg Thr               1005             - #   1010              - #  1015                         - - TTT GCC CTG GAG CGG AGA GGT TAC TCA GCC CG - #G CAT GAG GTC CGC CAG         3483                                                                       Phe Ala Leu Glu Arg Arg Gly Tyr Ser Ala Ar - #g His Glu Val Arg Gln           1020                1025 - #                1030 - #               1035        - - TTC CAT TTC ACA GCG TGG CCA GAG CAT GGT GT - #C CCC TAC CAC GCC ACG         3531                                                                       Phe His Phe Thr Ala Trp Pro Glu His Gly Va - #l Pro Tyr His Ala Thr                           1040 - #               1045  - #              1050             - - GGG CTG CTG GCC TTC ATC CGG CGT GTG AAG GC - #T TCC ACT CCA CCT GAT         3579                                                                       Gly Leu Leu Ala Phe Ile Arg Arg Val Lys Al - #a Ser Thr Pro Pro Asp                       1055     - #           1060      - #          1065                 - - GCC GGG CCC ATT GTC ATT CAC TGC AGT GCA GG - #A ACT GGC CGC ACA GGC         3627                                                                       Ala Gly Pro Ile Val Ile His Cys Ser Ala Gl - #y Thr Gly Arg Thr Gly                   1070         - #       1075          - #      1080                     - - TGC TAC ATC GTC CTG GAT GTG ATG CTG GAC AT - #G GCT GAA TGT GAG GGG         3675                                                                       Cys Tyr Ile Val Leu Asp Val Met Leu Asp Me - #t Ala Glu Cys Glu Gly               1085             - #   1090              - #  1095                         - - GTC GTG GAC ATT TAC AAC TGT GTG AAG ACC CT - #C TGT TCC CGA CGG GTC         3723                                                                       Val Val Asp Ile Tyr Asn Cys Val Lys Thr Le - #u Cys Ser Arg Arg Val           1100                1105 - #                1110 - #               1115        - - AAC ATG ATC CAG ACG GAG GAA CAA TAT ATC TT - #C ATC CAC GAT GCA ATC         3771                                                                       Asn Met Ile Gln Thr Glu Glu Gln Tyr Ile Ph - #e Ile His Asp Ala Ile                           1120 - #               1125  - #              1130             - - TTG GAG GCC TGC CTG TGT GGG GAG ACC ACC AT - #C CCT GTC AAC GAG TTC         3819                                                                       Leu Glu Ala Cys Leu Cys Gly Glu Thr Thr Il - #e Pro Val Asn Glu Phe                       1135     - #           1140      - #          1145                 - - AGG GCC ACC TAC AGG GAG ATG ATC CGC ATT GA - #C CCT CAG AGC AAT TCC         3867                                                                       Arg Ala Thr Tyr Arg Glu Met Ile Arg Ile As - #p Pro Gln Ser Asn Ser                   1150         - #       1155          - #      1160                     - - TCC CAG CTT CGG GAA GAG TTC CAG ACG CTG AA - #C TCG GTC ACG CCG CCG         3915                                                                       Ser Gln Leu Arg Glu Glu Phe Gln Thr Leu As - #n Ser Val Thr Pro Pro               1165             - #   1170              - #  1175                         - - CTG GAT GTG GAG GAG TGT AGC ATT GCC CTG CT - #G CCC CGG AAT CGA GAC         3963                                                                       Leu Asp Val Glu Glu Cys Ser Ile Ala Leu Le - #u Pro Arg Asn Arg Asp           1180                1185 - #                1190 - #               1195        - - AAG AAC CGT AGC ATG GAT GTG CTG CCA CCA GA - #C CGC YGC CTG CCC TTC         4011                                                                       Lys Asn Arg Ser Met Asp Val Leu Pro Pro As - #p Arg Xaa Leu Pro Phe                           1200 - #               1205  - #              1210             - - CTC ATC TCC AGT GAT GGG GAC CCC AAT AAC TA - #C ATC AAT GCA GCA CTG         4059                                                                       Leu Ile Ser Ser Asp Gly Asp Pro Asn Asn Ty - #r Ile Asn Ala Ala Leu                       1215     - #           1220      - #          1225                 - - ACT GAC AGC TAC ACA CGG AGC GCC GCC TTC AT - #C GTG ACC CTG CAC CCG         4107                                                                       Thr Asp Ser Tyr Thr Arg Ser Ala Ala Phe Il - #e Val Thr Leu His Pro                   1230         - #       1235          - #      1240                     - - CTG CAG AGT ACC ACG CCC GAC TTC TGG CGG CT - #G GTC TAC GAC TAC GGG         4155                                                                       Leu Gln Ser Thr Thr Pro Asp Phe Trp Arg Le - #u Val Tyr Asp Tyr Gly               1245             - #   1250              - #  1255                         - - TGC ACC TCC ATC GTC ATG CTG AAC CAA CTT AA - #C CAG TCC AAC TCC GCC         4203                                                                       Cys Thr Ser Ile Val Met Leu Asn Gln Leu As - #n Gln Ser Asn Ser Ala           1260                1265 - #                1270 - #               1275        - - TGG CCC TGC TTG CAG TAC TGG CCG GAG CCA GG - #C CGA CAG CAG TAT GGG         4251                                                                       Trp Pro Cys Leu Gln Tyr Trp Pro Glu Pro Gl - #y Arg Gln Gln Tyr Gly                           1280 - #               1285  - #              1290             - - CTC ATG GAG GTG GAG TTT GTG TCT GGC ACA GC - #A AAC GAG GAT TTG GTG         4299                                                                       Leu Met Glu Val Glu Phe Val Ser Gly Thr Al - #a Asn Glu Asp Leu Val                       1295     - #           1300      - #          1305                 - - TCC CGA GTG TTC CGG GTG CAG AAC TCT TCT CG - #G CTG CAG GAG GGT CAC         4347                                                                       Ser Arg Val Phe Arg Val Gln Asn Ser Ser Ar - #g Leu Gln Glu Gly His                   1310         - #       1315          - #      1320                     - - CTG CTG GTA CGG CAC TTC CAG TTT CTG CGT TG - #G TCT GCT TAT CGG GAC         4395                                                                       Leu Leu Val Arg His Phe Gln Phe Leu Arg Tr - #p Ser Ala Tyr Arg Asp               1325             - #   1330              - #  1335                         - - ACG CCT GAC TCC AGG AAG GCC TTT CTG CAC CT - #G TTG GCT GAG GTG GAC         4443                                                                       Thr Pro Asp Ser Arg Lys Ala Phe Leu His Le - #u Leu Ala Glu Val Asp           1340                1345 - #                1350 - #               1355        - - AAG TGG CAG GCA GAG AGT GGG GAT GGG CGC AC - #C GTG GTG CAT TGT CTC         4491                                                                       Lys Trp Gln Ala Glu Ser Gly Asp Gly Arg Th - #r Val Val His Cys Leu                           1360 - #               1365  - #              1370             - - AAC GGG GGT GGC CGC AGT GGC ACC TTC TGC GC - #C TGT GCC ACG GTC TTG         4539                                                                       Asn Gly Gly Gly Arg Ser Gly Thr Phe Cys Al - #a Cys Ala Thr Val Leu                       1375     - #           1380      - #          1385                 - - GAG ATG ATC CGC TGT CAC AGC CTG GTG GAT GT - #T TTC TTT GCT GCC AAA         4587                                                                       Glu Met Ile Arg Cys His Ser Leu Val Asp Va - #l Phe Phe Ala Ala Lys                   1390         - #       1395          - #      1400                     - - ACA CTT CGG AAC TAC AAG CCC AAT ATG GTG GA - #G ACC ATG GAT CAG TAT         4635                                                                       Thr Leu Arg Asn Tyr Lys Pro Asn Met Val Gl - #u Thr Met Asp Gln Tyr               1405             - #   1410              - #  1415                         - - CAT TTC TGC TAC GAC GTG GCC CTG GAG TAC CT - #G GAG GCT CTG GAG TTG         4683                                                                       His Phe Cys Tyr Asp Val Ala Leu Glu Tyr Le - #u Glu Ala Leu Glu Leu           1420                1425 - #                1430 - #               1435        - - AGA TAGCAGGCGC CTGACCTGGG GCACCCAGTG AACACCCAGG GCATGGCCC - #A              4736                                                                       Arg                                                                            - - TCATCCCAGA TGARGAGGGC CTGTGGCCCC AACTTTGCTC AGCCATAATT CC -             #ACAGGGAC   4796                                                                 - - AACACTGGAA CGGACGGACA CTGCACCATC TTGGTGACCC CCACGGGAAG GC -            #TGCAGGCC   4856                                                                 - - AAGGAGAAGC TTTGCAAGAC TGTATCAGCC CCACCTCTAG AGGGCCCTGC AG -            #ACCTGTGC   4916                                                                 - - AGAGAAGCTC GCCTGGACCA AAATAGCTAG TGCTGGAGAG CACAGGCCAG GC -            #CCCTCTGC   4976                                                                 - - TCCATCACAG TCCTTGGCCA GAAATGAATG AGTGTCTGCA GAGAGCACCC AT -            #GGTTTGCA   5036                                                                 - - CCCAGTATGG TCCTTTCTGC ACGTGGTGGA GGCTCACTGG GACTTGGCAG GG -            #GCTGAGTC   5096                                                                 - - CCCGAGAGTC CTGAAGCTGG GACTCTTCCC CGTCTCGCCG GTGGGACCCG CT -            #GAGCATCC   5156                                                                 - - TGCAGCTCCA TTCTCCATCC CCACTGCCCC TACAGACCTG GGGTGCTTTG CT -            #CGCTTTCC   5216                                                                 - - TCCTGCTTCT GAGCTTTTCC TGCAACAGGA CCCGTGCCTC CTTCCTGGGC TC -            #CATCCCTG   5276                                                                 - - CCTGGCCCAG TATATGCAGA ATGATATACT TCAGCTCCTT CTTCCCCTGG CC -            #TTTGGGTC   5336                                                                 - - TCCATGGTTC AGTCCTGCTC AGCTTGGGCC TGTGACAATC CACAAGGCTG AA -            #TCACAGCC   5396                                                                 - - CCTGGGGTTG AGGTCCCTGT GGCTCTTGGT GAGGCTGCCA CTGGATCGGG GC -            #AGGCTAGA   5456                                                                 - - ACAGGGCTGG TGTCAGCTCC TAGAGTACAG AGGAAGAAGG GATACTTTGG AA -            #TGGAGGAC   5516                                                                 - - CAGTGCTTTT TTTGTTGTTG TTATTTTGTT ATTTTTTTGA TGGGAGGGTG GG -            #AAGTTCTC   5576                                                                 - - TTTATAATGG GGTAGGCCAC ACCCCCATTT CGTGCCTCAA TTTCCCCATC TG -            #TAAACTGT   5636                                                                 - - AGATATGACT ACTGACCTAC CTCACAGGGG GCTGTGGGGA GGTGTAAGGT AA -            #TGTTTGTA   5696                                                                 - - AAGCGCTTTG TAAATAAATG TGCTCTCTGA ATGCCAAAAA AAAAAAAAAA AA -            #AAAAAAAA   5756                                                                 - - AAAAAAAAAA AAA              - #                  - #                      - #    5769                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1436 amino - #acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Ala Arg Ala Gln Ala Leu Val Leu Ala Le - #u Thr Phe Gln Phe Cys        1               5 - #                 10 - #                 15              - - Ala Pro Glu Thr Glu Thr Pro Ala Ala Gly Cy - #s Thr Phe Glu Glu Ala                   20     - #             25     - #             30                  - - Ser Asp Pro Val Val Pro Cys Glu Phe Ser Gl - #n Ala Gln Tyr Asp Asp               35         - #         40         - #         45                      - - Phe Gln Trp Glu Gln Val Arg Ile His Pro Gl - #y Thr Arg Thr Pro Glu           50             - #     55             - #     60                          - - Asp Leu Pro His Gly Ala Tyr Leu Met Val As - #n Ala Ser Gln His Thr       65                 - # 70                 - # 75                 - # 80       - - Pro Gly Gln Arg Ala His Ile Ile Phe Gln Th - #r Leu Ser Glu Asn Asp                       85 - #                 90 - #                 95              - - Thr His Cys Val Gln Phe Ser Tyr Phe Leu Ty - #r Ser Arg Asp Gly His                  100      - #           105      - #           110                  - - Ser Pro Gly Thr Leu Gly Val Tyr Val Arg Va - #l Asn Gly Gly Pro Leu              115          - #       120          - #       125                      - - Gly Ser Ala Val Trp Asn Met Thr Gly Ser Hi - #s Gly Arg Gln Trp His          130              - #   135              - #   140                          - - Gln Ala Glu Leu Ala Val Ser Thr Phe Trp Pr - #o Asn Glu Xaa Gln Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Phe Glu Ala Leu Ile Ser Pro Asp His Ly - #s Gly Tyr Ile Gly        Leu                                                                                             165  - #               170  - #               175             - - Asp Asp Ile Leu Leu Phe Ser Tyr Pro Cys Al - #a Lys Ala Pro His Phe                  180      - #           185      - #           190                  - - Ser Arg Leu Gly Asp Val Glu Val Asn Ala Gl - #y Gln Asn Ala Ser Phe              195          - #       200          - #       205                      - - Gln Cys Met Ala Ala Gly Arg Ala Ala Glu Al - #a Glu His Phe Phe Leu          210              - #   215              - #   220                          - - Gln Arg Gln Ser Gly Val Leu Val Pro Ala Al - #a Gly Val Arg His Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ser His Arg Arg Phe Leu Ala Thr Phe Pro Le - #u Ala Ser Val Gly        Arg                                                                                             245  - #               250  - #               255             - - Ser Glu Gln Asp Leu Tyr Arg Cys Val Ser Gl - #n Ala Pro Arg Gly Ala                  260      - #           265      - #           270                  - - Gly Val Ser Asn Phe Ala Glu Leu Ile Val Ly - #s Glu Pro Pro Thr Pro              275          - #       280          - #       285                      - - Ile Ala Pro Pro Gln Leu Leu Arg Ala Gly Pr - #o Thr Tyr Leu Ile Ile          290              - #   295              - #   300                          - - Gln Leu Asn Thr Asn Ser Ile Ile Gly Asp Gl - #y Pro Ile Val Arg Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Glu Ile Glu Tyr Arg Met Ala Arg Gly Pro Tr - #p Ala Glu Val His        Ala                                                                                             325  - #               330  - #               335             - - Val Asn Leu Xaa Thr Tyr Lys Leu Trp His Le - #u Asp Pro Asp Thr Glu                  340      - #           345      - #           350                  - - Tyr Glu Ile Ser Val Leu Leu Thr Arg Pro Gl - #y Asp Gly Gly Thr Gly              355          - #       360          - #       365                      - - Arg Pro Gly Pro Pro Leu Ile Ser Arg Thr Ly - #s Cys Ala Glu Pro Thr          370              - #   375              - #   380                          - - Arg Ala Pro Lys Gly Leu Ala Phe Ala Glu Il - #e Gln Ala Arg Gln Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Thr Leu Gln Trp Glu Pro Leu Gly Tyr Asn Va - #l Thr Arg Cys His        Thr                                                                                             405  - #               410  - #               415             - - Tyr Ala Val Ser Leu Cys Tyr Arg Tyr Thr Le - #u Gly Gly Ser His Asn                  420      - #           425      - #           430                  - - Gln Thr Ile Arg Glu Cys Val Lys Met Glu Ar - #g Gly Ala Ser Arg Tyr              435          - #       440          - #       445                      - - Thr Ile Lys Asn Leu Leu Pro Phe Arg Asn Il - #e His Val Arg Leu Ile          450              - #   455              - #   460                          - - Leu Thr Asn Pro Glu Gly Arg Lys Glu Gly Ly - #s Glu Val Thr Phe Gln      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Thr Asp Glu Asp Val Pro Gly Gly Ile Ala Al - #a Glu Ser Leu Thr        Phe                                                                                             485  - #               490  - #               495             - - Thr Pro Leu Glu Asp Met Ile Phe Leu Lys Tr - #p Glu Glu Pro Gln Glu                  500      - #           505      - #           510                  - - Pro Asn Gly Leu Ile Thr Gln Tyr Glu Ile Se - #r Tyr Gln Ser Ile Glu              515          - #       520          - #       525                      - - Ser Ser Asp Pro Ala Val Asn Val Pro Gly Pr - #o Arg Arg Thr Ile Ser          530              - #   535              - #   540                          - - Lys Leu Arg Asn Glu Thr Tyr His Val Phe Se - #r Asn Leu His Pro Gly      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Thr Thr Tyr Leu Phe Ser Val Arg Ala Arg Th - #r Ser Lys Gly Phe        Gly                                                                                             565  - #               570  - #               575             - - Gln Ala Ala Leu Thr Glu Ile Thr Thr Asn Il - #e Ser Ala Pro Ser Phe                  580      - #           585      - #           590                  - - Asp Tyr Ala Asp Met Pro Ser Pro Leu Gly Gl - #u Ser Glu Asn Thr Ile              595          - #       600          - #       605                      - - Thr Val Leu Leu Arg Pro Ala Gln Gly Arg Gl - #y Ala Pro Ile Ser Val          610              - #   615              - #   620                          - - Tyr Gln Val Val Val Glu Glu Glu Arg Pro Ar - #g Arg Leu Arg Arg Glu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Pro Gly Ala Gln Asp Cys Phe Ser Val Pro Le - #u Thr Phe Glu Thr        Ala                                                                                             645  - #               650  - #               655             - - Leu Ala Arg Gly Leu Val His Tyr Phe Gly Al - #a Glu Leu Ala Ala Ser                  660      - #           665      - #           670                  - - Ser Leu Leu Glu Ala Met Pro Phe Thr Val Gl - #y Asp Asn Gln Thr Tyr              675          - #       680          - #       685                      - - Arg Gly Phe Trp Asn Pro Pro Leu Glu Pro Ar - #g Lys Ala Tyr Leu Ile          690              - #   695              - #   700                          - - Tyr Phe Gln Ala Ala Ser His Leu Lys Gly Gl - #u Thr Arg Leu Asn Cys      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Ile Arg Ile Ala Arg Lys Ala Ala Cys Lys Gl - #u Ser Lys Arg Pro        Leu                                                                                             725  - #               730  - #               735             - - Glu Val Ser Gln Arg Ser Glu Glu Met Gly Le - #u Ile Leu Gly Ile Cys                  740      - #           745      - #           750                  - - Ala Gly Gly Leu Ala Val Leu Ile Leu Leu Le - #u Gly Ala Ile Ile Val              755          - #       760          - #       765                      - - Ile Ile Arg Lys Gly Lys Pro Val Asn Met Th - #r Lys Ala Thr Val Asn          770              - #   775              - #   780                          - - Tyr Arg Gln Glu Lys Thr His Met Met Ser Al - #a Val Asp Arg Ser Phe      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Thr Asp Gln Ser Thr Leu Gln Glu Asp Glu Ar - #g Leu Gly Leu Ser        Phe                                                                                             805  - #               810  - #               815             - - Met Asp Ala Pro Gly Tyr Ser Pro Arg Gly As - #p Gln Arg Ser Gly Gly                  820      - #           825      - #           830                  - - Val Thr Glu Ala Ser Ser Leu Leu Gly Gly Se - #r Pro Arg Arg Pro Cys              835          - #       840          - #       845                      - - Gly Arg Lys Gly Ser Pro Tyr His Thr Gly Gl - #n Leu His Pro Ala Val          850              - #   855              - #   860                          - - Arg Val Ala Asp Leu Leu Gln His Ile Asn Gl - #n Met Lys Thr Ala Glu      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Gly Tyr Gly Phe Lys Gln Glu Tyr Glu Ser Ph - #e Phe Glu Gly Trp        Asp                                                                                             885  - #               890  - #               895             - - Ala Thr Lys Lys Lys Asp Lys Leu Lys Gly Gl - #y Arg Gln Glu Pro Val                  900      - #           905      - #           910                  - - Ser Ala Tyr Asp Arg His His Val Lys Leu Hi - #s Pro Met Leu Ala Asp              915          - #       920          - #       925                      - - Pro Asp Ala Asp Tyr Ile Ser Ala Asn Tyr Il - #e Asp Gly Tyr His Arg          930              - #   935              - #   940                          - - Ser Asn His Phe Ile Ala Thr Gln Gly Pro Ly - #s Pro Glu Met Ile Tyr      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Asp Phe Trp Arg Met Val Trp Gln Glu Gln Cy - #s Ala Ser Ile Val        Met                                                                                             965  - #               970  - #               975             - - Ile Thr Lys Leu Val Glu Val Gly Arg Val Ly - #s Cys Ser Arg Tyr Trp                  980      - #           985      - #           990                  - - Pro Glu Asp Ser Asp Met Tyr Gly Asp Ile Ly - #s Ile Thr Leu Val Lys              995          - #       1000          - #      1005                     - - Thr Glu Thr Leu Ala Glu Tyr Val Val Arg Th - #r Phe Ala Leu Glu Arg          1010             - #   1015              - #  1020                         - - Arg Gly Tyr Ser Ala Arg His Glu Val Arg Gl - #n Phe His Phe Thr Ala      1025                1030 - #                1035 - #               1040        - - Trp Pro Glu His Gly Val Pro Tyr His Ala Th - #r Gly Leu Leu Ala Phe                      1045 - #               1050  - #              1055             - - Ile Arg Arg Val Lys Ala Ser Thr Pro Pro As - #p Ala Gly Pro Ile Val                  1060     - #           1065      - #          1070                 - - Ile His Cys Ser Ala Gly Thr Gly Arg Thr Gl - #y Cys Tyr Ile Val Leu              1075         - #       1080          - #      1085                     - - Asp Val Met Leu Asp Met Ala Glu Cys Glu Gl - #y Val Val Asp Ile Tyr          1090             - #   1095              - #  1100                         - - Asn Cys Val Lys Thr Leu Cys Ser Arg Arg Va - #l Asn Met Ile Gln Thr      1105                1110 - #                1115 - #               1120        - - Glu Glu Gln Tyr Ile Phe Ile His Asp Ala Il - #e Leu Glu Ala Cys Leu                      1125 - #               1130  - #              1135             - - Cys Gly Glu Thr Thr Ile Pro Val Asn Glu Ph - #e Arg Ala Thr Tyr Arg                  1140     - #           1145      - #          1150                 - - Glu Met Ile Arg Ile Asp Pro Gln Ser Asn Se - #r Ser Gln Leu Arg Glu              1155         - #       1160          - #      1165                     - - Glu Phe Gln Thr Leu Asn Ser Val Thr Pro Pr - #o Leu Asp Val Glu Glu          1170             - #   1175              - #  1180                         - - Cys Ser Ile Ala Leu Leu Pro Arg Asn Arg As - #p Lys Asn Arg Ser Met      1185                1190 - #                1195 - #               1200        - - Asp Val Leu Pro Pro Asp Arg Xaa Leu Pro Ph - #e Leu Ile Ser Ser Asp                      1205 - #               1210  - #              1215             - - Gly Asp Pro Asn Asn Tyr Ile Asn Ala Ala Le - #u Thr Asp Ser Tyr Thr                  1220     - #           1225      - #          1230                 - - Arg Ser Ala Ala Phe Ile Val Thr Leu His Pr - #o Leu Gln Ser Thr Thr              1235         - #       1240          - #      1245                     - - Pro Asp Phe Trp Arg Leu Val Tyr Asp Tyr Gl - #y Cys Thr Ser Ile Val          1250             - #   1255              - #  1260                         - - Met Leu Asn Gln Leu Asn Gln Ser Asn Ser Al - #a Trp Pro Cys Leu Gln      1265                1270 - #                1275 - #               1280        - - Tyr Trp Pro Glu Pro Gly Arg Gln Gln Tyr Gl - #y Leu Met Glu Val Glu                      1285 - #               1290  - #              1295             - - Phe Val Ser Gly Thr Ala Asn Glu Asp Leu Va - #l Ser Arg Val Phe Arg                  1300     - #           1305      - #          1310                 - - Val Gln Asn Ser Ser Arg Leu Gln Glu Gly Hi - #s Leu Leu Val Arg His              1315         - #       1320          - #      1325                     - - Phe Gln Phe Leu Arg Trp Ser Ala Tyr Arg As - #p Thr Pro Asp Ser Arg          1330             - #   1335              - #  1340                         - - Lys Ala Phe Leu His Leu Leu Ala Glu Val As - #p Lys Trp Gln Ala Glu      1345                1350 - #                1355 - #               1360        - - Ser Gly Asp Gly Arg Thr Val Val His Cys Le - #u Asn Gly Gly Gly Arg                      1365 - #               1370  - #              1375             - - Ser Gly Thr Phe Cys Ala Cys Ala Thr Val Le - #u Glu Met Ile Arg Cys                  1380     - #           1385      - #          1390                 - - His Ser Leu Val Asp Val Phe Phe Ala Ala Ly - #s Thr Leu Arg Asn Tyr              1395         - #       1400          - #      1405                     - - Lys Pro Asn Met Val Glu Thr Met Asp Gln Ty - #r His Phe Cys Tyr Asp          1410             - #   1415              - #  1420                         - - Val Ala Leu Glu Tyr Leu Glu Ala Leu Glu Le - #u Arg                      1425                1430 - #                1435                               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1457 amino - #acids                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Asp Val Ala Ala Ala Ala Leu Pro Ala Ph - #e Val Ala Leu Trp Leu      1               5   - #                10  - #                15               - - Leu Tyr Pro Trp Pro Leu Leu Gly Ser Ala Le - #u Gly Gln Phe Ser Ala                  20      - #            25      - #            30                   - - Gly Gly Cys Thr Phe Asp Asp Gly Pro Gly Al - #a Cys Asp Tyr His Gln              35          - #        40          - #        45                       - - Asp Leu Tyr Asp Asp Phe Glu Trp Val His Va - #l Ser Ala Gln Glu Pro          50              - #    55              - #    60                           - - His Tyr Leu Pro Pro Glu Met Pro Gln Gly Se - #r Tyr Met Val Val Asp      65                  - #70                  - #75                  - #80        - - Ser Ser Asn His Asp Pro Gly Glu Lys Ala Ar - #g Leu Gln Leu Pro Thr                      85  - #                90  - #                95               - - Met Lys Glu Asn Asp Thr His Cys Ile Asp Ph - #e Ser Tyr Leu Leu Tyr                  100      - #           105      - #           110                  - - Ser Gln Lys Gly Leu Asn Pro Gly Thr Leu As - #n Ile Leu Val Arg Val              115          - #       120          - #       125                      - - Asn Lys Gly Pro Leu Ala Asn Pro Ile Trp As - #n Val Thr Gly Phe Thr          130              - #   135              - #   140                          - - Gly Arg Asp Trp Leu Arg Ala Glu Leu Ala Va - #l Ser Thr Phe Trp Pro      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asn Glu Tyr Gln Val Ile Phe Glu Ala Glu Va - #l Ser Gly Gly Arg        Ser                                                                                             165  - #               170  - #               175             - - Gly Tyr Ile Ala Ile Asp Asp Ile Gln Val Le - #u Ser Tyr Pro Cys Asp                  180      - #           185      - #           190                  - - Lys Ser Pro His Phe Leu Arg Leu Gly Asp Va - #l Glu Val Asn Ala Gly              195          - #       200          - #       205                      - - Gln Asn Ala Thr Phe Gln Cys Ile Ala Thr Gl - #y Arg Asp Ala Val His          210              - #   215              - #   220                          - - Asn Lys Leu Trp Leu Gln Arg Arg Asn Gly Gl - #u Asp Ile Pro Val Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Thr Lys Asn Ile Asn His Arg Arg Phe Al - #a Ala Ser Phe Arg        Leu                                                                                             245  - #               250  - #               255             - - Gln Glu Val Thr Lys Thr Asp Gln Asp Leu Ty - #r Arg Cys Val Thr Gln                  260      - #           265      - #           270                  - - Ser Glu Arg Gly Ser Gly Val Ser Asn Phe Al - #a Gln Leu Ile Val Arg              275          - #       280          - #       285                      - - Glu Pro Pro Arg Pro Ile Ala Pro Pro Gln Le - #u Leu Gly Val Gly Pro          290              - #   295              - #   300                          - - Thr Tyr Leu Leu Ile Gln Leu Asn Ala Asn Se - #r Ile Ile Gly Asp Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Ile Ile Leu Lys Glu Val Glu Tyr Arg Me - #t Thr Ser Gly Ser        Trp                                                                                             325  - #               330  - #               335             - - Thr Glu Thr His Ala Val Asn Ala Pro Thr Ty - #r Lys Leu Trp His Leu                  340      - #           345      - #           350                  - - Asp Pro Asp Thr Glu Tyr Glu Ile Arg Val Le - #u Leu Thr Arg Pro Gly              355          - #       360          - #       365                      - - Glu Gly Gly Thr Gly Leu Pro Gly Pro Pro Le - #u Ile Thr Arg Thr Lys          370              - #   375              - #   380                          - - Cys Ala Glu Pro Met Arg Thr Pro Lys Thr Le - #u Lys Ile Ala Glu Ile      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gln Ala Arg Arg Ile Ala Val Asp Trp Glu Se - #r Leu Gly Tyr Asn        Ile                                                                                             405  - #               410  - #               415             - - Thr Arg Cys His Thr Phe Asn Val Thr Ile Cy - #s Tyr His Tyr Phe Arg                  420      - #           425      - #           430                  - - Gly His Asn Glu Ser Arg Ala Asp Cys Leu As - #p Met Asp Pro Lys Ala              435          - #       440          - #       445                      - - Pro Gln His Val Val Asn His Leu Pro Pro Ty - #r Thr Asn Val Ser Leu          450              - #   455              - #   460                          - - Lys Met Ile Leu Thr Asn Pro Glu Gly Arg Ly - #s Glu Ser Glu Glu Thr      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Ile Ile Gln Thr Asp Glu Asp Val Pro Gly Pr - #o Val Pro Val Lys        Ser                                                                                             485  - #               490  - #               495             - - Leu Gln Gly Thr Ser Phe Glu Asn Lys Ile Ph - #e Leu Asn Trp Lys Glu                  500      - #           505      - #           510                  - - Pro Leu Glu Pro Asn Gly Ile Ile Thr Gln Ty - #r Glu Val Ser Tyr Ser              515          - #       520          - #       525                      - - Ser Ile Arg Ser Phe Asp Pro Ala Val Pro Va - #l Ala Gly Pro Pro Gln          530              - #   535              - #   540                          - - Thr Val Ser Asn Leu Trp Asn Ser Thr His Hi - #s Val Phe Met His Leu      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - His Pro Gly Thr Thr Tyr Gln Phe Phe Ile Ar - #g Ala Ser Thr Val        Lys                                                                                             565  - #               570  - #               575             - - Gly Phe Gly Pro Ala Thr Ala Ile Asn Val Th - #r Thr Asn Ile Ser Ala                  580      - #           585      - #           590                  - - Pro Ser Leu Pro Asp Tyr Glu Gly Val Asp Al - #a Ser Leu Asn Glu Thr              595          - #       600          - #       605                      - - Ala Thr Thr Ile Thr Val Leu Leu Arg Pro Al - #a Gln Ala Lys Gly Ala          610              - #   615              - #   620                          - - Pro Ile Ser Ala Tyr Gln Ile Val Val Glu Gl - #n Leu His Pro His Arg      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Thr Lys Arg Glu Ala Gly Ala Met Glu Cys Ty - #r Gln Val Pro Val        Thr                                                                                             645  - #               650  - #               655             - - Tyr Gln Asn Ala Leu Ser Gly Gly Ala Pro Ty - #r Tyr Phe Ala Ala Glu                  660      - #           665      - #           670                  - - Leu Pro Pro Gly Asn Leu Pro Glu Pro Ala Pr - #o Phe Thr Val Gly Asp              675          - #       680          - #       685                      - - Asn Arg Thr Tyr Lys Gly Phe Trp Asn Pro Pr - #o Leu Ala Pro Arg Lys          690              - #   695              - #   700                          - - Gly Tyr Asn Ile Tyr Phe Gln Ala Met Ser Se - #r Val Glu Lys Glu Thr      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Lys Thr Gln Cys Val Arg Ile Ala Thr Lys Al - #a Ala Ala Thr Glu        Glu                                                                                             725  - #               730  - #               735             - - Pro Glu Val Ile Pro Asp Pro Ala Lys Gln Th - #r Asp Arg Val Val Lys                  740      - #           745      - #           750                  - - Ile Ala Gly Ile Ser Ala Gly Ile Leu Val Ph - #e Ile Leu Leu Leu Leu              755          - #       760          - #       765                      - - Val Val Ile Val Ile Val Lys Lys Ser Lys Le - #u Ala Lys Lys Arg Lys          770              - #   775              - #   780                          - - Asp Ala Met Gly Asn Thr Arg Gln Glu Met Th - #r His Met Val Asn Ala      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Met Asp Arg Ser Tyr Ala Asp Gln Ser Thr Le - #u His Ala Glu Asp        Pro                                                                                             805  - #               810  - #               815             - - Leu Ser Leu Thr Phe Met Asp Gln His Asn Ph - #e Ser Pro Arg Leu Pro                  820      - #           825      - #           830                  - - Asn Asp Pro Leu Val Pro Thr Ala Val Leu As - #p Glu Asn His Ser Ala              835          - #       840          - #       845                      - - Thr Ala Glu Ser Ser Arg Leu Leu Asp Val Pr - #o Arg Tyr Leu Cys Glu          850              - #   855              - #   860                          - - Gly Thr Glu Ser Pro Tyr Gln Thr Gly Gln Le - #u His Pro Ala Ile Arg      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Val Ala Asp Leu Leu Gln His Ile Asn Leu Me - #t Lys Thr Ser Asp        Ser                                                                                             885  - #               890  - #               895             - - Tyr Gly Phe Lys Glu Glu Tyr Glu Ser Phe Ph - #e Glu Gly Gln Ser Ala                  900      - #           905      - #           910                  - - Ser Trp Asp Val Ala Lys Lys Asp Gln Asn Ar - #g Ala Lys Asn Arg Tyr              915          - #       920          - #       925                      - - Gly Asn Ile Ile Ala Tyr Asp His Ser Arg Va - #l Ile Leu Gln Pro Val          930              - #   935              - #   940                          - - Glu Asp Asp Pro Ser Ser Asp Tyr Ile Asn Al - #a Asn Tyr Ile Asp Ile      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Trp Leu Tyr Arg Asp Gly Tyr Gln Arg Pro Se - #r His Tyr Ile Ala        Thr                                                                                             965  - #               970  - #               975             - - Gln Gly Pro Val His Glu Thr Val Tyr Asp Ph - #e Trp Arg Met Val Trp                  980      - #           985      - #           990                  - - Gln Glu Gln Ser Ala Cys Ile Val Met Val Th - #r Asn Leu Val Glu Val              995          - #       1000          - #      1005                     - - Gly Arg Val Lys Cys Tyr Lys Tyr Trp Pro As - #p Asp Thr Glu Val Tyr          1010             - #   1015              - #  1020                         - - Gly Asp Phe Lys Val Thr Cys Val Glu Met Gl - #u Pro Leu Ala Glu Tyr      1025                1030 - #                1035 - #               1040        - - Val Val Arg Thr Phe Thr Leu Glu Arg Arg Gl - #y Tyr Asn Glu Ile Arg                      1045 - #               1050  - #              1055             - - Glu Val Lys Gln Phe His Phe Thr Gly Trp Pr - #o Asp His Gly Val Pro                  1060     - #           1065      - #          1070                 - - Tyr His Ala Thr Gly Leu Leu Ser Phe Ile Ar - #g Arg Val Lys Leu Ser              1075         - #       1080          - #      1085                     - - Asn Pro Pro Ser Ala Gly Pro Ile Val Val Hi - #s Cys Ser Ala Gly Ala          1090             - #   1095              - #  1100                         - - Gly Arg Thr Gly Cys Tyr Ile Val Ile Asp Il - #e Met Leu Asp Met Ala      1105                1110 - #                1115 - #               1120        - - Glu Arg Glu Gly Val Val Asp Ile Tyr Asn Cy - #s Val Lys Ala Leu Arg                      1125 - #               1130  - #              1135             - - Ser Arg Arg Ile Asn Met Val Gln Thr Glu Gl - #u Gln Tyr Ile Phe Ile                  1140     - #           1145      - #          1150                 - - His Asp Ala Ile Leu Glu Ala Cys Leu Cys Gl - #y Glu Thr Ala Ile Pro              1155         - #       1160          - #      1165                     - - Val Cys Glu Phe Lys Ala Ala Tyr Phe Asp Me - #t Ile Arg Ile Asp Ser          1170             - #   1175              - #  1180                         - - Gln Thr Asn Ser Ser His Leu Lys Asp Glu Ph - #e Gln Thr Leu Asn Ser      1185                1190 - #                1195 - #               1200        - - Val Thr Pro Arg Leu Gln Ala Glu Asp Cys Se - #r Ile Ala Cys Leu Pro                      1205 - #               1210  - #              1215             - - Arg Asn His Asp Lys Asn Arg Phe Met Asp Me - #t Leu Pro Pro Asp Arg                  1220     - #           1225      - #          1230                 - - Cys Leu Pro Phe Leu Ile Thr Ile Asp Gly Gl - #u Ser Ser Asn Tyr Ile              1235         - #       1240          - #      1245                     - - Asn Ala Ala Leu Met Asp Ser Tyr Arg Gln Pr - #o Ala Ala Phe Ile Val          1250             - #   1255              - #  1260                         - - Thr Gln Tyr Pro Leu Pro Asn Thr Val Lys As - #p Phe Trp Arg Leu Val      1265                1270 - #                1275 - #               1280        - - Tyr Asp Tyr Gly Cys Thr Ser Ile Val Met Le - #u Asn Glu Val Asp Leu                      1285 - #               1290  - #              1295             - - Ser Gln Gly Cys Pro Gln Tyr Trp Pro Glu Gl - #u Gly Met Leu Arg Tyr                  1300     - #           1305      - #          1310                 - - Gly Pro Ile Gln Val Glu Cys Met Ser Cys Se - #r Met Asp Cys Asp Val              1315         - #       1320          - #      1325                     - - Ile Asn Arg Ile Phe Arg Ile Cys Asn Leu Th - #r Arg Pro Gln Glu Gly          1330             - #   1335              - #  1340                         - - Tyr Leu Met Val Gln Gln Phe Gln Tyr Leu Gl - #y Trp Ala Ser His Arg      1345                1350 - #                1355 - #               1360        - - Glu Val Pro Gly Ser Lys Arg Ser Phe Leu Ly - #s Leu Ile Leu Gln Val                      1365 - #               1370  - #              1375             - - Glu Lys Trp Gln Glu Glu Cys Glu Glu Gly Gl - #u Gly Arg Thr Ile Ile                  1380     - #           1385      - #          1390                 - - His Cys Leu Asn Gly Gly Gly Arg Ser Gly Me - #t Phe Cys Ala Ile Gly              1395         - #       1400          - #      1405                     - - Ile Val Val Glu Met Val Lys Arg Gln Asn Va - #l Val Asp Val Phe His          1410             - #   1415              - #  1420                         - - Ala Val Lys Thr Leu Arg Asn Ser Lys Pro As - #n Met Val Glu Ala Pro      1425                1430 - #                1435 - #               1440        - - Glu Gln Tyr Arg Phe Cys Tyr Asp Val Ala Le - #u Glu Tyr Leu Glu Ser                      1445 - #               1450  - #              1455             - - Ser                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1452 amino - #acids                                               (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Arg Thr Leu Gly Thr Cys Leu Val Thr Le - #u Ala Gly Leu Leu Leu      1               5   - #                10  - #                15               - - Thr Ala Ala Gly Glu Thr Phe Ser Gly Gly Cy - #s Leu Phe Asp Glu Pro                  20      - #            25      - #            30                   - - Tyr Ser Thr Cys Gly Tyr Ser Gln Ala Asp Gl - #u Asp Asp Phe Asn Trp              35          - #        40          - #        45                       - - Glu Gln Val Asn Thr Leu Thr Lys Pro Thr Se - #r Asp Pro Trp Met Pro          50              - #    55              - #    60                           - - Ser Gly Ser Phe Met Leu Val Asn Thr Ser Gl - #y Lys Pro Glu Gly Gln      65                  - #70                  - #75                  - #80        - - Arg Ala His Leu Leu Leu Pro Gln Leu Lys Gl - #u Asn Asp Thr His Cys                      85  - #                90  - #                95               - - Ile Asp Phe His Tyr Phe Val Ser Ser Lys Se - #r Asn Ala Ala Pro Gly                  100      - #           105      - #           110                  - - Leu Leu Asn Val Tyr Val Lys Val Asn Asn Gl - #y Pro Leu Gly Asn Pro              115          - #       120          - #       125                      - - Ile Trp Asn Ile Ser Gly Asp Pro Thr Arg Th - #r Trp His Arg Ala Glu          130              - #   135              - #   140                          - - Leu Ala Ile Ser Thr Phe Trp Pro Asn Phe Ty - #r Gln Val Ile Phe Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Val Thr Ser Gly His Gln Gly Tyr Leu Al - #a Ile Asp Glu Val        Lys                                                                                             165  - #               170  - #               175             - - Val Leu Gly His Pro Cys Thr Arg Thr Pro Hi - #s Phe Leu Arg Ile Gln                  180      - #           185      - #           190                  - - Asn Val Glu Val Asn Ala Gly Gln Phe Ala Th - #r Phe Gln Cys Ser Ala              195          - #       200          - #       205                      - - Ile Gly Arg Thr Val Ala Gly Asp Arg Leu Tr - #p Leu Gln Gly Ile Asp          210              - #   215              - #   220                          - - Val Arg Asp Ala Pro Leu Lys Glu Ile Lys Va - #l Thr Ser Ser Arg Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Phe Ile Ala Ser Phe Asn Val Val Asn Thr Th - #r Lys Arg Asp Ala        Gly                                                                                             245  - #               250  - #               255             - - Lys Tyr Arg Cys Met Ile Cys Thr Glu Gly Gl - #y Val Gly Ile Ser Asn                  260      - #           265      - #           270                  - - Tyr Ala Glu Leu Val Val Lys Glu Pro Pro Va - #l Pro Ile Ala Pro Pro              275          - #       280          - #       285                      - - Gln Leu Ala Ser Val Gly Ala Thr Tyr Leu Tr - #p Ile Gln Leu Asn Ala          290              - #   295              - #   300                          - - Asn Ser Ile Asn Gly Asp Gly Pro Ile Val Al - #a Arg Glu Val Glu Tyr      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Cys Thr Ala Ser Gly Ser Trp Asn Asp Arg Gl - #n Pro Val Asp Ser        Thr                                                                                             325  - #               330  - #               335             - - Ser Tyr Lys Ile Gly His Leu Asp Pro Asp Th - #r Glu Tyr Glu Ile Ser                  340      - #           345      - #           350                  - - Val Leu Leu Thr Arg Pro Gly Glu Gly Gly Th - #r Gly Ser Pro Gly Pro              355          - #       360          - #       365                      - - Ala Leu Arg Thr Arg Thr Lys Cys Ala Asp Pr - #o Met Arg Gly Pro Arg          370              - #   375              - #   380                          - - Lys Leu Glu Val Val Glu Val Lys Ser Arg Gl - #n Ile Thr Ile Arg Trp      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Pro Phe Gly Tyr Asn Val Thr Arg Cys Hi - #s Ser Tyr Asn Leu        Thr                                                                                             405  - #               410  - #               415             - - Val His Tyr Gly Tyr Gln Val Gly Gly Gln Gl - #u Gln Val Arg Glu Glu                  420      - #           425      - #           430                  - - Val Ser Trp Asp Thr Asp Asn Ser His Pro Gl - #n His Thr Ile Thr Asn              435          - #       440          - #       445                      - - Leu Ser Pro Tyr Thr Asn Val Ser Val Lys Le - #u Ile Leu Met Asn Pro          450              - #   455              - #   460                          - - Glu Gly Arg Lys Glu Ser Gln Glu Leu Thr Va - #l Gln Thr Asp Glu Asp      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Leu Pro Gly Ala Val Pro Thr Glu Ser Ile Gl - #n Gly Ser Ala Phe        Glu                                                                                             485  - #               490  - #               495             - - Glu Lys Ile Phe Leu Gln Trp Arg Glu Pro Th - #r Gln Thr Tyr Gly Val                  500      - #           505      - #           510                  - - Ile Thr Leu Tyr Glu Ile Thr Tyr Lys Ala Va - #l Ser Ser Phe Asp Pro              515          - #       520          - #       525                      - - Glu Ile Asp Leu Ser Asn Gln Ser Gly Arg Va - #l Ser Lys Leu Gly Asn          530              - #   535              - #   540                          - - Glu Thr His Phe Leu Phe Phe Gly Leu Tyr Pr - #o Gly Thr Thr Tyr Ser      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Phe Thr Ile Arg Ala Ser Thr Ala Lys Gly Ph - #e Gly Pro Pro Ala        Thr                                                                                             565  - #               570  - #               575             - - Asn Gln Phe Thr Thr Lys Ile Ser Ala Pro Se - #r Met Pro Ala Tyr Glu                  580      - #           585      - #           590                  - - Phe Glu Thr Pro Leu Asn Gln Thr Asp Asn Th - #r Val Thr Val Met Leu              595          - #       600          - #       605                      - - Lys Pro Ala Gln Ser Arg Gly Ala Pro Val Se - #r Val Tyr Gln Ile Val          610              - #   615              - #   620                          - - Val Glu Glu Glu Arg Pro Arg Arg Thr Lys Ly - #s Thr Thr Glu Ile Leu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Lys Cys Tyr Pro Val Pro Ile His Phe Gln As - #n Ala Ser Ile Leu        Asn                                                                                             645  - #               650  - #               655             - - Ser Gln Tyr Tyr Phe Ala Ala Glu Phe Pro Al - #a Asp Ser Leu Gln Ala                  660      - #           665      - #           670                  - - Ala Gln Pro Phe Thr Ile Gly Asp Asn Lys Th - #r Tyr Asn Gly Tyr Trp              675          - #       680          - #       685                      - - Asn Thr Pro Leu Leu Pro His Lys Ser Tyr Ar - #g Ile Tyr Tyr Gln Ala          690              - #   695              - #   700                          - - Ala Ser Arg Ala Asn Gly Glu Thr Lys Ile As - #p Cys Val Arg Val Ala      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Thr Lys Gly Ala Val Thr Pro Lys Pro Val Pr - #o Glu Pro Glu Lys        Gln                                                                                             725  - #               730  - #               735             - - Thr Asp His Thr Val Lys Ile Ala Gly Val Il - #e Ala Gly Ile Leu Leu                  740      - #           745      - #           750                  - - Phe Val Ile Ile Phe Leu Gly Val Val Leu Va - #l Met Lys Lys Arg Lys              755          - #       760          - #       765                      - - Leu Ala Lys Lys Arg Lys Glu Thr Met Ser Se - #r Thr Arg Gln Glu Met          770              - #   775              - #   780                          - - Thr Val Met Val Asn Ser Met Asp Lys Ser Ty - #r Ala Glu Gln Gly Thr      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Asn Cys Asp Glu Ala Phe Ser Phe Met Gly Th - #r His Asn Leu Asn        Gly                                                                                             805  - #               810  - #               815             - - Arg Ser Val Ser Ser Pro Ser Ser Phe Thr Me - #t Lys Thr Asn Thr Leu                  820      - #           825      - #           830                  - - Ser Thr Ser Val Pro Asn Ser Tyr Tyr Pro As - #p Glu Thr His Thr Met              835          - #       840          - #       845                      - - Ala Ser Asp Thr Ser Ser Leu Ala Gln Pro Hi - #s Thr Tyr Lys Lys Arg          850              - #   855              - #   860                          - - Glu Ala Ala Asp Val Pro Tyr Gln Thr Gly Gl - #n Leu His Pro Ala Ile      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Arg Val Ala Asp Leu Leu Gln His Ile Thr Gl - #n Met Lys Cys Ala        Glu                                                                                             885  - #               890  - #               895             - - Gly Tyr Gly Phe Lys Glu Glu Tyr Glu Ser Ph - #e Phe Glu Gly Gln Ser                  900      - #           905      - #           910                  - - Ala Pro Trp Asp Ser Ala Lys Lys Asp Glu As - #n Arg Met Lys Asn Arg              915          - #       920          - #       925                      - - Tyr Gly Asn Ile Ile Ala Tyr Asp His Ser Ar - #g Val Arg Leu Gln Met          930              - #   935              - #   940                          - - Leu Glu Gly Asp Asn Asn Ser Asp Tyr Ile As - #n Gly Asn Tyr Ile Asp      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Gly Tyr His Arg Pro Asn His Tyr Ile Ala Th - #r Gln Gly Pro Met        Gln                                                                                             965  - #               970  - #               975             - - Glu Thr Ile Tyr Asp Phe Trp Arg Met Val Tr - #p His Glu Asn Thr Ala                  980      - #           985      - #           990                  - - Ser Ile Ile Met Val Thr Asn Leu Val Glu Va - #l Gly Arg Val Lys Cys              995          - #       1000          - #      1005                     - - Cys Lys Tyr Trp Pro Asp Asp Thr Glu Ile Ty - #r Lys Asp Ile Lys Val          1010             - #   1015              - #  1020                         - - Thr Leu Ile Asp Thr Glu Leu Leu Ala Glu Ty - #r Val Ile Arg Thr Phe      1025                1030 - #                1035 - #               1040        - - Ala Val Glu Lys Arg Gly Ile His Glu Ile Ar - #g Glu Ile Arg Gln Phe                      1045 - #               1050  - #              1055             - - His Phe Thr Gly Trp Pro Asp His Gly Val Pr - #o Tyr His Ala Thr Gly                  1060     - #           1065      - #          1070                 - - Leu Leu Gly Phe Val Arg Gln Val Lys Ser Ly - #s Ser Pro Pro Asn Ala              1075         - #       1080          - #      1085                     - - Gly Pro Leu Val Val His Cys Ser Ala Gly Al - #a Gly Arg Thr Gly Cys          1090             - #   1095              - #  1100                         - - Phe Ile Val Ile Asp Ile Met Leu Asp Met Al - #a Glu Arg Glu Gly Val      1105                1110 - #                1115 - #               1120        - - Val Asp Ile Tyr Asn Cys Val Arg Glu Leu Ar - #g Ser Arg Arg Val Asn                      1125 - #               1130  - #              1135             - - Met Val Gln Thr Glu Glu Gln Tyr Val Phe Il - #e His Asp Ala Ile Leu                  1140     - #           1145      - #          1150                 - - Glu Ala Cys Leu Cys Gly Asp Thr Ser Ile Pr - #o Ala Ser Gln Val Arg              1155         - #       1160          - #      1165                     - - Ser Leu Tyr Tyr Asp Met Asn Lys Leu Asp Pr - #o Gln Thr Asn Ser Ser          1170             - #   1175              - #  1180                         - - Gln Ile Lys Glu Glu Phe Arg Thr Leu Asn Me - #t Val Thr Pro Thr Leu      1185                1190 - #                1195 - #               1200        - - Arg Val Glu Asp Cys Ser Ile Ala Leu Leu Pr - #o Arg Asn His Glu Lys                      1205 - #               1210  - #              1215             - - Asn Arg Cys Met Asp Ile Leu Pro Pro Asp Ar - #g Cys Leu Pro Phe Leu                  1220     - #           1225      - #          1230                 - - Ile Thr Ile Asp Gly Glu Ser Ser Asn Tyr Il - #e Asn Ala Ala Leu Met              1235         - #       1240          - #      1245                     - - Asp Ser Tyr Lys Gln Pro Ser Ala Phe Ile Va - #l Thr Gln His Pro Leu          1250             - #   1255              - #  1260                         - - Pro Asn Thr Val Lys Asp Phe Trp Arg Leu Va - #l Leu Asp Tyr His Cys      1265                1270 - #                1275 - #               1280        - - Thr Ser Val Val Met Leu Asn Asp Val Asp Pr - #o Ala Gln Leu Cys Pro                      1285 - #               1290  - #              1295             - - Gln Tyr Trp Pro Glu Asn Gly Val His Arg Hi - #s Gly Pro Ile Gln Val                  1300     - #           1305      - #          1310                 - - Glu Phe Val Ser Ala Asp Leu Glu Glu Asp Il - #e Ile Ser Arg Ile Phe              1315         - #       1320          - #      1325                     - - Arg Ile Tyr Asn Ala Ser Arg Pro Gln Asp Gl - #y His Arg Met Val Gln          1330             - #   1335              - #  1340                         - - Gln Phe Gln Phe Leu Gly Trp Pro Met Tyr Ar - #g Asp Thr Pro Val Ser      1345                1350 - #                1355 - #               1360        - - Lys Arg Ser Phe Leu Lys Leu Ile Arg Gln Va - #l Asp Lys Trp Gln Glu                      1365 - #               1370  - #              1375             - - Glu Tyr Asn Gly Gly Glu Gly Pro Thr Val Va - #l His Cys Leu Asn Gly                  1380     - #           1385      - #          1390                 - - Gly Gly Arg Ser Gly Thr Phe Cys Ala Ile Se - #r Ile Val Cys Glu Met              1395         - #       1400          - #      1405                     - - Leu Arg His Gln Arg Thr Val Asp Val Phe Hi - #s Ala Val Lys Thr Leu          1410             - #   1415              - #  1420                         - - Arg Asn Asn Lys Pro Asn Met Val Asp Leu Le - #u Asp Gln Tyr Lys Phe      1425                1430 - #                1435 - #               1440        - - Cys Tyr Glu Val Ala Leu Glu Tyr Leu Asn Se - #r Gly                                      1445 - #               1450                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Active-site                                                     (B) LOCATION: 1..2                                                            (D) OTHER INFORMATION: - #/note= "Let 'X' located at position                      1 represe - #nt either Histidine or Aspartic Acid"              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 6..7                                                            (D) OTHER INFORMATION: - #/note= "Let 'X' located at position                      6 represe - #nt either Isoleucine or Valine."                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Xaa Phe Trp Arg Met Xaa Trp                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 11..12                                                          (D) OTHER INFORMATION: - #/note= "Let the 'N' at position 11                       represent - #Inosine."                                          - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 17..18                                                          (D) OTHER INFORMATION: - #/note= "Let the 'N' at position 17                       represent - #Inosine."                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AYTTYTGGMG NATGRTNTGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4..5                                                            (D) OTHER INFORMATION: - #/note= "Let 'X' located at position                      4 represe - #nt either Phenyalanine or Histidine."              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Trp Pro Asp Xaa Gly Val Pro                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 3..4                                                            (D) OTHER INFORMATION: - #/note= "Let 'N' located at position                      3 represe - #nt Inosine."                                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..13                                                          (D) OTHER INFORMATION: - #/note= "Let 'N' located at position                      12 repres - #ent Inosine."                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GGNACRWRRT CNGGCCA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Ty - #r                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Glu Gly Pro Trp Leu Glu Glu Glu Glu Glu Al - #a Tyr                      1               5   - #                10                                    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule encoding areceptor protein tyrosine phosphatase λ polypeptide whichdephosphorylates phosphorylated tyrosine residues, said nucleic acidmolecule comprising:(a) a nucleotide sequence comprising nucleotides379-4686 of the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1); (b) anucleotide sequence which encodes the amino acid sequence shown in FIG.1 (SEQ ID NO:2); and (c) a nucleotide sequence which hybridizes understringent conditions to the nucleotide sequence of (a) or (b).
 2. Theisolated nucleic acid molecule according to claim 1 which encodes ahuman receptor protein tyrosine phosphatase λ polypeptide.
 3. Theisolated nucleic acid molecule according to claim 1 which encodes amurine receptor protein tyrosine phosphatase λ polypeptide.
 4. Theisolated nucleic acid molecule according to claim 1 which comprisesnucleotides 379-4686 of the nucleotide sequence shown in FIG. 1 (SEQ IDNO:1).
 5. The isolated nucleic acid molecule according to claim 1 whichcomprises a nucleotide sequence which encodes the amino acid sequenceshown in FIG. 1 (SEQ ID NO:2).
 6. A vector comprising the nucleic acidmolecule according to any one of claims 1, 2, 3, 4 or 5 operably linkedto control sequences recognized by a host cell transformed with thevector.
 7. A host cell transformed with the vector according to claim 6.8. A process for producing a receptor protein tyrosine phosphatase λpolypeptide which dephosphorylates phosphorylated tyrosine residues,said process comprising culturing the transformed host cell of claim 7and recovering said polypeptide from the cell culture.